Dottorato di ricerca in Chimica

UNIVERSITA DEGLI STUDI DI SALERNO FACOLTA DI SCIENZE MATEMATICHE FISICHE E NATURALI

Synthesis and properties of linear and cyclic peptoids

-X Cycle- Nuova serie (2008-2011)

Tutor Prof Francesco De Riccardis PhD candidate Chiara De Cola Co-tutor Prof Irene Izzo Coordinatore Prof Gaetano Guerra

1

INDEX

CHAPTER 1 INTRODUCTION 3 11 PEPTIDOMIMETICS 5 12 PEPTOIDS A PROMISING CLASS OF PEPTIDOMIMETICS 9 13 CONFORMATIONAL STUDIES OF PEPTOIDS 11 14 PEPTOIDSrsquo APPLICATIONS 14 15 PEPTOID SINTHESYS 39 16 SYNTHESYS OF PNA MONOMERS AND OLIGOMERS 41 17 AIMS OF THE WORK 49 CHAPTER 2 CARBOXYALKYL PEPTOID PNAS SYNTHESIS AND HYBRIDIZATION PROPERTIES 51 21 INTRODUCTION 51 22 RESULTS AND DISCUSSION 55 23 CONCLUSIONS 60 24 EXPERIMENTAL SECTION 60 CHAPTER 3 STRUCTURAL ANALYSIS OF CYCLOPEPTOIDS AND THEIR COMPLEXES 80 31 INTRODUCTION 80 32 RESULTS AND DISCUSSION 85 33 CONCLUSIONS 102 34 EXPERIMENTAL SECTION 103 CHAPTER 4 CATIONIC CYCLOPEPTOIDS AS POTENTIAL MACROCYCLIC NONVIRAL VECTORS 115 41 INTRODUCTION 115 42 RESULTS AND DISCUSSION 122 43 CONCLUSIONS 125 44 EXPERIMENTAL SECTION 125 CHAPTER 5 COMPLEXATION WITH GD(III) OF CARBOXYETHYL CYCLOPEPTOIDS AS POSSIBLE CONTRAST AGENTS

IN MRI 132 51 INTRODUCTION 132 52 LARIAT ETHER AND CLICK CHEMISTRY 135 53 RESULTS AND DISCUSSION 141 54 EXPERIMENTAL SECTION 145 CHAPTER 6 CYCLOPEPTOIDS AS MIMETIC OF NATURAL DEFENSINS 157 61 INTRODUCTION 157 62 RESULTS AND DISCUSSION 162 63 CONCLUSIONS 167 65 EXPERIMENTAL SECTION 167

2

List of abbreviations

Cbz Benzyl chloroformate

DCC NNrsquo-Dicyclohexylcarbodiimide

DCM Dichloromethane

DIPEA Diisopropylethylamine

DMF N Nrsquo-dimethylformamide

Fmoc Fluorenylmethyloxycarbonyl chloride

HBTU O-Benzotriazole-NNNN-tetramethyl-uronium-hexafluorophosphate

HATU O-(7-Azabenzotriazol-1-yl)-NNNN-tetramethyluronium hexafluorophosphate

PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate

PNA Peptide nucleic acid

t-Bu terz-Butyl

THF Tetrahydrofuran

3

Chapter 1

1 Introduction

ldquoGiunto a questo punto della vita quale chimico davanti alla tabella del Sistema Periodico o agli indici

monumentali del Beilstein o del Landolt non vi ravvisa sparsi i tristi brandelli o i trofei del proprio passato

professionale Non ha che da sfogliare un qualsiasi trattato e le memorie sorgono a grappoli crsquoegrave fra noi chi ha

legato il suo destino indelebilmente al bromo o al propilene o al gruppo ndashNCO o allrsquoacido glutammico ed ogni

studente in chimica davanti ad un qualsiasi trattato dovrebbe essere consapevole che in una di quelle pagine forse in

una sola riga o formula o parola sta scritto il suo avvenire in caratteri indecifrabili ma che diventeranno chiari

ltltPOIgtgt dopo il successo o lrsquoerrore o la colpa la vittoria o la disfatta

Ogni chimico non piugrave giovane riaprendo alla pagina ltlt verhangnisvoll gtgt quel medesimo trattato egrave percosso

da amore o disgusto si rallegra o si disperardquo

Da ldquoIl Sistema Periodicordquo Primo Levi

Proteins are vital for essentially every known organism The development of a deeper understanding

of proteinndashprotein interactions and the design of novel peptides which selectively interact with proteins

are fields of active research

One way how nature controls the protein functions within living cells is by regulating proteinndash

protein interactions These interactions exist on nearly every level of cellular function which means they

are of key importance for virtually every process in a living organism Regulation of the protein-protein

interactions plays a crucial role in unicellular and multicellular organisms including man and

represents the perfect example of molecular recognition1

Synthetic methods like the solid-phase peptide synthesis (SPPS) developed by B Merrifield2 made it

possible to synthesize polypeptides for pharmacological and clinical testing as well as for use as drugs

or in diagnostics

As a result different new peptide-based drugs are at present accessible for the treatment of prostate

and breast cancer as HIV protease inhibitors or as ACE inhibitors to treat hypertension and congestive

heart failures to mention only few examples1

Unfortunately these small peptides typically show high conformational flexibility and a low in-vivo

stability which hampers their application as tools in medicinal diagnostics or molecular biology A

major difficulty in these studies is the conformational flexibility of most peptides and the high

dependence of their conformations on the surrounding environment which often leads to a

conformational equilibrium The high flexibility of natural polypeptides is due to the multiple

conformations that are energetically possible for each residue of the incorporated amino acids Every

amino acid has two degrees of conformational freedom NndashCα (Φ) and CαndashCO (Ψ) resulting in

approximately 9 stable local conformations1 For a small peptide with only 40 amino acids in length the

1 A Grauer B Koumlnig Eur J Org Chem 2009 5099ndash5111

2 a) R B Merrifield Federation Proc 1962 21 412 b) R B Merrifield J Am Chem Soc 1964 86 2149ndash2154

4

number of possible conformations which need to be considered escalates to nearly 10403 This

extraordinary high flexibility of natural amino acids leads to the fact that short polypeptides consisting

of the 20 proteinogenic amino acids rarely form any stable 3D structures in solution1 There are only

few examples reported in the literature where short to medium-sized peptides (lt30ndash50 amino acids)

were able to form stable structures In most cases they exist in aqueous solution in numerous

dynamically interconverting conformations Moreover the number of stable short peptide structures

which are available is very limited because of the need to use amino acids having a strong structure

inducing effect like for example helix-inducing amino acids as leucine glutamic acid or lysine In

addition it is dubious whether the solid state conformations determined by X-ray analysis are identical

to those occurring in solution or during the interactions of proteins with each other1 Despite their wide

range of important bioactivities polypeptides are generally poor drugs Typically they are rapidly

degraded by proteases in vivo and are frequently immunogenic

This fact has inspired prevalent efforts to develop peptide mimics for biomedical applications a task

that presents formidable challenges in molecular design

11 Peptidomimetics

One very versatile strategy to overcome such drawbacks is the use of peptidomimetics4 These are

small molecules which mimic natural peptides or proteins and thus produce the same biological effects

as their natural role models

They also often show a decreased activity in comparison to the protein from which they are derived

These mimetics should have the ability to bind to their natural targets in the same way as the natural

peptide sequences from which their structure was derived do and should produce the same biological

effects It is possible to design these molecules in such a way that they show the same biological effects

as their peptide role models but with enhanced properties like a higher proteolytic stability higher

bioavailability and also often with improved selectivity or potency This makes them interesting targets

for the discovery of new drug candidates

For the progress of potent peptidomimetics it is required to understand the forces that lead to

proteinndashprotein interactions with nanomolar or often even higher affinities

These strong interactions between peptides and their corresponding proteins are mainly based on side

chain interactions indicating that the peptide backbone itself is not an absolute requirement for high

affinities

This allows chemists to design peptidomimetics basically from any scaffold known in chemistry by

replacing the amide backbone partially or completely by other structures Peptidomimetics furthermore

can have some peculiar qualities such as a good solubility in aqueous solutions access to facile

sequences-specific assembly of monomers containing chemically diverse side chains and the capacity to

form stable biomimetic folded structures5

Most important is that the backbone is able to place the amino acid side chains in a defined 3D-

position to allow interactions with the target protein too Therefore it is necessary to develop an idea of

the required structure of the peptidomimetic to show a high activity against its biological target

3 J Venkatraman S C Shankaramma P Balaram Chem Rev 2001 101 3131ndash3152 4 J A Patch K Kirshenbaum S L Seurynck R N Zuckermann and A E Barron in Pseudo-peptides in Drug

Development ed P E Nielsen Wiley-VCH Weinheim Germany 2004 1ndash31

5

The most significant parameters for an optimal peptidomimetics are stereochemistry charge and

hydrophobicity and these parameters can be examined by systematic exchange of single amino acids

with modified amino acid As a result the key residues which are essential for the biological activity

can be identified As next step the 3D arrangement of these key residues needs to be analyzed by the use

of compounds with rigid conformations to identify the most active structure1 In general the

development of peptidomimetics is based mainly on the knowledge of the electronic conformational

and topochemical properties of the native peptide to its target

Two structural factors are particularly important for the synthesis of peptidomimetics with high

biological activity firstly the mimetic has to have a convenient fit to the binding site and secondly the

functional groups polar and hydrophobic regions of the mimetic need to be placed in defined positions

to allow the useful interactions to take place1

One very successful approach to overcome these drawbacks is the introduction of conformational

constraints into the peptide sequence This can be done for example by the incorporation of amino acids

which can only adopt a very limited number of different conformations or by cyclisation (main chain to

main chain side chain to main chain or side chain to side chain)5

Peptidomimetics furthermore can contain two different modifications amino acid modifications or

peptideslsquo backbone modifications

Figure 11 reports the most important ways to modify the backbone of peptides at different positions

Figure 11 Some of the more common modifications to the peptide backbone (adapted from

literature)6

5a) C Toniolo M Goodman Introduction to the Synthesis of Peptidomimetics in Methods of Organic Chemistry

Synthesis of Peptides and Peptidomimetics (Ed M Goodman) Thieme Stuttgart New York 2003 vol E22c p

1ndash2 b) D J Hill M J Mio R B Prince T S Hughes J S Moore Chem Rev 2001 101 3893ndash4012 6 J Gante Angew Chem Int Ed Engl 1994 33 1699ndash1720

6

Backbone peptides modifications are a method for synthesize optimal peptidomimetics in particular

is possible

the replacement of the α-CH group by nitrogen to form azapeptides

the change from amide to ester bond to get depsipeptides

the exchange of the carbonyl function by a CH2 group

the extension of the backbone (β-amino acids and γ-amino acids)

the amide bond inversion (a retro-inverse peptidomimetic)

The carba alkene or hydroxyethylene groups are used in exchange for the amide bond

The shift of the alkyl group from α-CH group to α-N group

Most of these modifications do not guide to a higher restriction of the global conformations but they

have influence on the secondary structure due to the altered intramolecular interactions like different

hydrogen bonding Additionally the length of the backbone can be different and a higher proteolytic

stability occurs in most cases 1

12 Peptoids A Promising Class of Peptidomimetics

If we shift the chain of α-CH group by one position on the peptide backbone we produced the

disappearance of all the intra-chain stereogenic centers and the formation of a sequence of variously

substituted N-alkylglycines (figure 12)

Figure 12 Comparison of a portion of a peptide chain with a portion of a peptoid chain

Oligomers of N-substituted glycine or peptoids were developed by Zuckermann and co-workers in

the early 1990lsquos7 They were initially proposed as an accessible class of molecules from which lead

compounds could be identified for drug discovery

Peptoids can be described as mimics of α-peptides in which the side chain is attached to the

backbone amide nitrogen instead of the α-carbon (figure 12) These oligomers are an attractive scaffold

for biological applications because they can be generated using a straightforward modular synthesis that

allows the incorporation of a wide variety of functionalities8 Peptoids have been evaluated as tools to

7 R J Simon R S Kania R N Zuckermann V D Huebner D A Jewell S Banville S Ng LWang S

Rosenberg C K Marlowe D C Spellmeyer R Tan A D Frankel D V Santi F E Cohen and P A Bartlett

Proc Natl Acad Sci U S A 1992 89 9367ndash9371

7

study biomolecular interactions8 and also hold significant promise for therapeutic applications due to

their enhanced proteolytic stabilities8 and increased cellular permeabilities

9 relative to α-peptides

Biologically active peptoids have also been discovered by rational design (ie using molecular

modeling) and were synthesized either individually or in parallel focused libraries10

For some

applications a well-defined structure is also necessary for peptoid function to display the functionality

in a particular orientation or to adopt a conformation that promotes interaction with other molecules

However in other biological applications peptoids lacking defined structures appear to possess superior

activities over structured peptoids

This introduction will focus primarily on the relationship between peptoid structure and function A

comprehensive review of peptoids in drug discovery detailing peptoid synthesis biological

applications and structural studies was published by Barron Kirshenbaum Zuckermann and co-

workers in 20044 Since then significant advances have been made in these areas and new applications

for peptoids have emerged In addition new peptoid secondary structural motifs have been reported as

well as strategies to stabilize those structures Lastly the emergence of peptoid with tertiary structures

has driven chemists towards new structures with peculiar properties and side chains Peptoid monomers

are linked through polyimide bonds in contrast to the amide bonds of peptides Unfortunately peptoids

do not have the hydrogen of the peptide secondary amide and are consequently incapable of forming

the same types of hydrogen bond networks that stabilize peptide helices and β-sheets

The peptoids oligomers backbone is achiral however stereogenic centers can be included in the side

chains to obtain secondary structures with a preferred handedness4 In addition peptoids carrying N-

substituted versions of the proteinogenic side chains are highly resistant to degradation by proteases

which is an important attribute of a pharmacologically useful peptide mimic4

13 Conformational studies of peptoids

The fact that peptoids are able to form a variety of secondary structural elements including helices

and hairpin turns suggests a range of possible conformations that can allow the generation of functional

folds11

Some studies of molecular mechanics have demonstrated that peptoid oligomers bearing bulky

chiral (S)-N-(1-phenylethyl) side chains would adopt a polyproline type I helical conformation in

agreement with subsequent experimental findings12

Kirshenbaum at al12 has shown agreement between theoretical models and the trans amide of N-

aryl peptoids and suggested that they may form polyproline type II helices Combined these studies

suggest that the backbone conformational propensities evident at the local level may be readily

translated into the conformations of larger oligomers chains

N-α-chiral side chains were shown to promote the folding of these structures in both solution and the

solid state despite the lack of main chain chirality and secondary amide hydrogen bond donors crucial

to the formation of many α-peptide secondary structures

8 S M Miller R J Simon S Ng R N Zuckermann J M Kerr W H Moos Bioorg Med Chem Lett 1994 4

2657ndash2662 9 Y UKwon and T Kodadek J Am Chem Soc 2007 129 1508ndash1509 10 T Hara S R Durell M C Myers and D H Appella J Am Chem Soc 2006 128 1995ndash2004 11 G L Butterfoss P D Renfrew B Kuhlman K Kirshenbaum R Bonneau J Am Chem Soc 2009 131

16798ndash16807

8

While computational studies initially suggested that steric interactions between N-α-chiral aromatic

side chains and the peptoid backbone primarily dictated helix formation both intra- and intermolecular

aromatic stacking interactions12

have also been proposed to participate in stabilizing such helices13

In addition to this consideration Gorske et al14

selected side chain functionalities to look at the

effects of four key types of noncovalent interactions on peptoid amide cistrans equilibrium (1) nrarrπ

interactions between an amide and an aromatic ring (nrarrπAr) (2) nrarrπ interactions between two

carbonyls (nrarrπ C=O) (3) side chain-backbone steric interactions and (4) side chain-backbone

hydrogen bonding interactions In figure 13 are reported as example only nrarrπAr and nrarrπC=O

interactions

A B

Figure 13 A (Left) nrarrπAr interaction (indicated by the red arrow) proposed to increase of

Kcistrans (equilibrium constant between cis and trans conformation) for peptoid backbone amides (Right)

Newman projection depicting the nrarrπAr interaction B (Left) nrarrπC=O interaction (indicated by

the red arrow) proposed to reduce Kcistrans for the donating amide in peptoids (Right) Newman

projection depicting the nrarrπC=O interaction

Other classes of peptoid side chains have been designed to introduce dipole-dipole hydrogen

bonding and electrostatic interactions stabilizing the peptoid helix

In addition such constraints may further rigidify peptoid structure potentially increasing the ability

of peptoid sequences for selective molecular recognition

In a relatively recent contribution Kirshenbaum15

reported that peptoids undergo to a very efficient

head-to-tail cyclisation using standard coupling agents The introduction of the covalent constraint

enforces conformational ordering thus facilitating the crystallization of a cyclic peptoid hexamer and a

cyclic peptoid octamer

Peptoids can form well-defined three-dimensional folds in solution too In fact peptoid oligomers

with α-chiral side chains were shown to adopt helical structures 16

a threaded loop structure was formed

12 C W Wu T J Sanborn R N Zuckermann A E Barron J Am Chem Soc 2001 123 2958ndash2963 13 T J Sanborn C W Wu R N Zuckermann A E Barron Biopolymers 2002 63 12ndash20 14

B C Gorske J R Stringer B L Bastian S A Fowler H E Blackwell J Am Chem Soc 2009 131

16555ndash16567 15 S B Y Shin B Yoo L J Todaro K Kirshenbaum J Am Chem Soc 2007 129 3218-3225 16 (a) K Kirshenbaum A E Barron R A Goldsmith P Armand E K Bradley K T V Truong K A Dill F E

Cohen R N Zuckermann Proc Natl Acad Sci USA 1998 95 4303ndash4308 (b) P Armand K Kirshenbaum R

A Goldsmith S Farr-Jones A E Barron K T V Truong K A Dill D F Mierke F E Cohen R N

Zuckermann E K Bradley Proc Natl Acad Sci USA 1998 95 4309ndash4314 (c) C W Wu K Kirshenbaum T

J Sanborn J A Patch K Huang K A Dill R N Zuckermann A E Barron J Am Chem Soc 2003 125

13525ndash13530

9

by intramolecular hydrogen bonds in peptoid nonamers20

head-to-tail macrocyclizations provided

conformationally restricted cyclic peptoids

These studies demonstrate the importance of (1) access to chemically diverse monomer units and (2)

precise control of secondary structures to expand applications of peptoid helices

The degree of helical structure increases as chain length grows and for these oligomers becomes

fully developed at length of approximately 13 residues Aromatic side chain-containing peptoid helices

generally give rise to CD spectra that are strongly reminiscent of that of a peptide α-helix while peptoid

helices based on aliphatic groups give rise to a CD spectrum that resembles the polyproline type-I

helical

14 Peptoidsrsquo Applications

The well-defined helical structure associated with appropriately substituted peptoid oligomers can be

employed to construct compounds that closely mimic the structures and functions of certain bioactive

peptides In this paragraph are shown some examples of peptoids that have antibacterial and

antimicrobial properties molecular recognition properties of metal complexing peptoids of catalytic

peptoids and of peptoids tagged with nucleobases

141 Antibacterial and antimicrobial properties

The antibiotic activities of structurally diverse sets of peptidespeptoids derive from their action on

microbial cytoplasmic membranes The model proposed by ShaindashMatsuzakindashHuan17

(SMH) presumes

alteration and permeabilization of the phospholipid bilayer with irreversible damage of the critical

membrane functions Cyclization of linear peptidepeptoid precursors (as a mean to obtain

conformational order) has been often neglected18

despite the fact that nature offers a vast assortment of

powerful cyclic antimicrobial peptides19

However macrocyclization of N-substituted glycines gives

17 (a) Matsuzaki K Biochim Biophys Acta 1999 1462 1 (b) Yang L Weiss T M Lehrer R I Huang H W

Biophys J 2000 79 2002 (c) Shai Y BiochimBiophys Acta 1999 1462 55 18 Chongsiriwatana N P Patch J A Czyzewski A M Dohm M T Ivankin A Gidalevitz D Zuckermann

R N Barron A E Proc Natl Acad Sci USA 2008 105 2794 19 Interesting examples are (a) Motiei L Rahimipour S Thayer D A Wong C H Ghadiri M R Chem

Commun 2009 3693 (b) Fletcher J T Finlay J A Callow J A Ghadiri M R Chem Eur J 2007 13 4008

(c) Au V S Bremner J B Coates J Keller P A Pyne S G Tetrahedron 2006 62 9373 (d) Fernandez-

Lopez S Kim H-S Choi E C Delgado M Granja J R Khasanov A Kraehenbuehl K Long G

Weinberger D A Wilcoxen K M Ghadiri M R Nature 2001 412 452 (e) Casnati A Fabbi M Pellizzi N

Pochini A Sansone F Ungaro R Di Modugno E Tarzia G Bioorg Med Chem Lett 1996 6 2699 (f)

Robinson J A Shankaramma C S Jetter P Kienzl U Schwendener R A Vrijbloed J W Obrecht D

Bioorg Med Chem 2005 13 2055

10

circular peptoids20

showing reduced conformational freedom21

and excellent membrane-permeabilizing

activity22

Antimicrobial peptides (AMPs) are found in myriad organisms and are highly effective against

bacterial infections23

The mechanism of action for most AMPs is permeabilization of the bacterial

cytoplasmic membrane which is facilitated by their amphipathic structure24

The cationic region of AMPs confers a degree of selectivity for the membranes of bacterial cells over

mammalian cells which have negatively charged and neutral membranes respectively The

hydrophobic portions of AMPs are supposed to mediate insertion into the bacterial cell membrane

Although AMPs possess many positive attributes they have not been developed as drugs due to the

poor pharmacokinetics of α-peptides This problem creates an opportunity to develop peptoid mimics of

AMPs as antibiotics and has sparked considerable research in this area25

De Riccardis26

et al investigated the antimicrobial activities of five new cyclic cationic hexameric α-

peptoids comparing their efficacy with the linear cationic and the cyclic neutral counterparts (figure

14)

20 (a) Craik D J Cemazar M Daly N L Curr Opin Drug Discovery Dev 2007 10 176 (b) Trabi M Craik

D J Trend Biochem Sci 2002 27 132 21 (a) Maulucci N Izzo I Bifulco G Aliberti A De Cola C Comegna D Gaeta C Napolitano A Pizza

C Tedesco C Flot D De Riccardis F Chem Commun 2008 3927 (b) Kwon Y-U Kodadek T Chem

Commun 2008 5704 (c) Vercillo O E Andrade C K Z Wessjohann L A Org Lett 2008 10 205 (d) Vaz

B Brunsveld L Org Biomol Chem 2008 6 2988 (e) Wessjohann L A Andrade C K Z Vercillo O E

Rivera D G In Targets in Heterocyclic Systems Attanasi O A Spinelli D Eds Italian Society of Chemistry

2007 Vol 10 pp 24ndash53 (f) Shin S B Y Yoo B Todaro L J Kirshenbaum K J Am Chem Soc 2007 129

3218 (g) Hioki H Kinami H Yoshida A Kojima A Kodama M Taraoka S Ueda K Katsu T

Tetrahedron Lett 2004 45 1091 22 (a) Chatterjee J Mierke D Kessler H Chem Eur J 2008 14 1508 (b) Chatterjee J Mierke D Kessler

H J Am Chem Soc 2006 128 15164 (c) Nnanabu E Burgess K Org Lett 2006 8 1259 (d) Sutton P W

Bradley A Farragraves J Romea P Urpigrave F Vilarrasa J Tetrahedron 2000 56 7947 (e) Sutton P W Bradley

A Elsegood M R Farragraves J Jackson R F W Romea P Urpigrave F Vilarrasa J Tetrahedron Lett 1999 40

2629 23 A Peschel and H-G Sahl Nat Rev Microbiol 2006 4 529ndash536 24 R E W Hancock and H-G Sahl Nat Biotechnol 2006 24 1551ndash1557 25 For a review of antimicrobial peptoids see I Masip E Pegraverez Payagrave A Messeguer Comb Chem High

Throughput Screen 2005 8 235ndash239 26 D Comegna M Benincasa R Gennaro I Izzo F De Riccardis Bioorg Med Chem 2010 18 2010ndash2018

11

Figure 14 Structures of synthesized linear and cyclic peptoids described by De Riccardis at al Bn

= benzyl group Boc= t-butoxycarbonyl group

The synthesized peptoids have been assayed against clinically relevant bacteria and fungi including

Escherichia coli Staphylococcus aureus amphotericin β-resistant Candida albicans and Cryptococcus

neoformans27

The purpose of this study was to explore the biological effects of the cyclisation on positively

charged oligomeric N-alkylglycines with the idea to mimic the natural amphiphilic peptide antibiotics

The long-term aim of the effort was to find a key for the rational design of novel antimicrobial

compounds using the finely tunable peptoid backbone

The exploration for possible biological activities of linear and cyclic α-peptoids was started with the

assessment of the antimicrobial activity of the known21a

N-benzyloxyethyl cyclohomohexamer (Figure

14 Block I) This neutral cyclic peptoid was considered a promising candidate in the antimicrobial

27 M Benincasa M Scocchi S Pacor A Tossi D Nobili G Basaglia M Busetti R J Gennaro Antimicrob

Chemother 2006 58 950

12

assays for its high affinity to the first group alkali metals (Ka ~ 106 for Na+ Li

+ and K

+)

21a and its ability

to promote Na+H

+ transmembrane exchange through ion-carrier mechanism

28 a behavior similar to that

observed for valinomycin a well known K+-carrier with powerful antibiotic activity

29 However

determination of the MIC values showed that neutral chains did not exert any antimicrobial activity

against a group of selected pathogenic fungi and of Gram-negative and Gram-positive bacterial strains

even at concentrations up to 1 mM

Detailed structurendashactivity relationship (SAR) studies30

have revealed that the amphiphilicity of the

peptidespeptidomimetics and the total number of positively charged residues impact significantly on

the antimicrobial activity Therefore cationic versions of the neutral cyclic α-peptoids were planned

(Figure 14 block I and block II compounds) In this study were also included the linear cationic

precursors to evaluate the effect of macrocyclization on the antimicrobial activity Cationic peptoids

were tested against four pathogenic fungi and three clinically relevant bacterial strains The tests showed

a marked increase of the antibacterial and antifungal activities with cyclization The presence of charged

amino groups also influenced the antimicrobial efficacy as shown by the activity of the bi- and

tricationic compounds when compared with the ineffective neutral peptoid These results are the first

indication that cyclic peptoids can represent new motifs on which to base artificial antibiotics

In 2003 Barron and Patch31

reported peptoid mimics of the helical antimicrobial peptide magainin-2

that had low micromolar activity against Escherichia coli (MIC = 5ndash20 mM) and Bacillus subtilis (MIC

= 1ndash5 mM)

The magainins exhibit highly selective and potent antimicrobial activity against a broad spectrum of

organisms5 As these peptides are facially amphipathic the magainins have a cationic helical face

mostly composed of lysine residues as well as hydrophobic aromatic (phenylalanine) and hydrophobic

aliphatic (valine leucine and isoleucine) helical faces This structure is responsible for their activity4

Peptoids have been shown to form remarkably stable helices with physical characteristics similar to

those of peptide polyproline type-I helices In fact a series of peptoid magainin mimics with this type

of three-residue periodic sequences has been synthesized4 and tested against E coli JM109 and B

subtulis BR151 In all cases peptoids are individually more active against the Gram-positive species

The amount of hemolysis induced by these peptoids correlated well with their hydrophobicity In

summary these recently obtain results demonstrate that certain amphipathic peptoid sequences are also

capable of antibacterial activity

142 Molecular Recognition

Peptoids are currently being studied for their potential to serve as pharmaceutical agents and as

chemical tools to study complex biomolecular interactions Peptoidndashprotein interactions were first

demonstrated in a 1994 report by Zuckermann and co-workers8 where the authors examined the high-

affinity binding of peptoid dimers and trimers to G-protein-coupled receptors These groundbreaking

studies have led to the identification of several peptoids with moderate to good affinity and more

28 C De Cola S Licen D Comegna E Cafaro G Bifulco I Izzo P Tecilla F De Riccardis Org Biomol

Chem 2009 7 2851 29 N R Clement J M Gould Biochemistry 1981 20 1539 30 J I Kourie A A Shorthouse Am J Physiol Cell Physiol 2000 278 C1063 31 J A Patch and A E Barron J Am Chem Soc 2003 125 12092ndash 12093

13

importantly excellent selectivity for protein targets that implicated in a range of human diseases There

are many different interactions between peptoid and protein and these interactions can induce a certain

inhibition cellular uptake and delivery Synthetic molecules capable of activating the expression of

specific genes would be valuable for the study of biological phenomena and could be therapeutically

useful From a library of ~100000 peptoid hexamers Kodadek and co-workers recently identified three

peptoids (24-26) with low micromolar binding affinities for the coactivator CREB-binding protein

(CBP) in vitro (Figure 15)9 This coactivator protein is involved in the transcription of a large number

of mammalian genes and served as a target for the isolation of peptoid activation domain mimics Of

the three peptoids only 24 was selective for CBP while peptoids 25 and 26 showed higher affinities for

bovine serum albumin The authors concluded that the promiscuous binding of 25 and 26 could be

attributed to their relatively ―sticky natures (ie aromatic hydrophobic amide side chains)

Inhibitors of proteasome function that can intercept proteins targeted for degradation would be

valuable as both research tools and therapeutic agents In 2007 Kodadek and co-workers32

identified the

first chemical modulator of the proteasome 19S regulatory particle (which is part of the 26S proteasome

an approximately 25 MDa multi-catalytic protease complex responsible for most non-lysosomal protein

degradation in eukaryotic cells) A ―one bead one compound peptoid library was constructed by split

and pool synthesis

Figure 15 Peptoid hexamers 24 25 and 26 reported by Kodadek and co-workers and their

dissociation constants (KD) for coactivator CBP33

Peptoid 24 was able to function as a transcriptional

activation domain mimic (EC50 = 8 mM)

32 H S Lim C T Archer T Kodadek J Am Chem Soc 2007 129 7750

14

Each peptoid molecule was capped with a purine analogue in hope of biasing the library toward

targeting one of the ATPases which are part of the 19S regulatory particle Approximately 100 000

beads were used in the screen and a purine-capped peptoid heptamer (27 Figure 16) was identified as

the first chemical modulator of the 19S regulatory particle In an effort to evidence the pharmacophore

of 2733

(by performing a ―glycine scan similar to the ―alanine scan in peptides) it was shown that just

the core tetrapeptoid was necessary for the activity

Interestingly the synthesis of the shorter peptoid 27 gave in the experiments made on cells a 3- to

5-fold increase in activity relative to 28 The higher activity in the cell-based essay was likely due to

increased cellular uptake as 27 does not contain charged residues

Figure 16 Purine capped peptoid heptamer (28) and tetramer (27) reported by Kodadek preventing

protein degradation

143 Metal Complexing Peptoids

A desirable attribute for biomimetic peptoids is the ability to show binding towards receptor sites

This property can be evoked by proper backbone folding due to

1) local side-chain stereoelectronic influences

2) coordination with metallic species

3) presence of hydrogen-bond donoracceptor patterns

Those three factors can strongly influence the peptoidslsquo secondary structure which is difficult to

observe due to the lack of the intra-chain C=OHndashN bonds present in the parent peptides

Most peptoidslsquo activities derive by relatively unstructured oligomers If we want to mimic the

sophisticated functions of proteins we need to be able to form defined peptoid tertiary structure folds

and introduce functional side chains at defined locations Peptoid oligomers can be already folded into

helical secondary structures They can be readily generated by incorporating bulky chiral side chains

33 HS Lim C T Archer Y C Kim T Hutchens T Kodadek Chem Commun 2008 1064

15

into the oligomer2234-35

Such helical secondary structures are extremely stable to chemical denaturants

and temperature13

The unusual stability of the helical structure may be a consequence of the steric

hindrance of backbone φ angle by the bulky chiral side chains36

Zuckermann and co-workers synthesized biomimetic peptoids with zinc-binding sites8 since zinc-

binding motifs in protein are well known Zinc typically stabilizes native protein structures or acts as a

cofactor for enzyme catalysis37-38

Zinc also binds to cellular cysteine-rich metallothioneins solely for

storage and distribution39

The binding of zinc is typically mediated by cysteines and histidines

50-51 In

order to create a zinc-binding site they incorporated thiol and imidazole side chains into a peptoid two-

helix bundle

Classic zinc-binding motifs present in proteins and including thiol and imidazole moieties were

aligned in two helical peptoid sequences in a way that they could form a binding site Fluorescence

resonance energy transfer (FRET) reporter groups were located at the edge of this biomimetic structure

in order to measure the distance between the two helical segments and probe and at the same time the

zinc binding propensity (29 Figure 17)

29

Figure 17 Chemical structure of 29 one of the twelve folded peptoids synthesized by Zuckermann

able to form a Zn2+

complex

Folding of the two helix bundles was allowed by a Gly-Gly-Pro-Gly middle region The study

demonstrated that certain peptoids were selective zinc binders at nanomolar concentration

The formation of the tertiary structure in these peptoids is governed by the docking of preorganized

peptoid helices as shown in these studies40

A survey of the structurally diverse ionophores demonstrated that the cyclic arrangement represents a

common archetype equally promoted by chemical design22f

and evolutionary pressure Stereoelectronic

effects caused by N- (and C-) substitution22f

andor by cyclisation dictate the conformational ordering of

peptoidslsquo achiral polyimide backbone In particular the prediction and the assessment of the covalent

34 Wu C W Kirshenbaum K Sanborn T J Patch J A Huang K Dill K A Zuckermann R N Barron A

E J Am Chem Soc 2003 125 13525ndash13530 35 Armand P Kirshenbaum K Falicov A Dunbrack R L Jr DillK A Zuckermann R N Cohen F E

Folding Des 1997 2 369ndash375 36 K Kirshenbaum R N Zuckermann K A Dill Curr Opin Struct Biol 1999 9 530ndash535 37 Coleman J E Annu ReV Biochem 1992 61 897ndash946 38 Berg J M Godwin H A Annu ReV Biophys Biomol Struct 1997 26 357ndash371 39 Cousins R J Liuzzi J P Lichten L A J Biol Chem 2006 281 24085ndash24089 40 B C Lee R N Zuckermann K A Dill J Am Chem Soc 2005 127 10999ndash11009

16

constraints induced by macrolactamization appears crucial for the design of conformationally restricted

peptoid templates as preorganized synthetic scaffolds or receptors In 2008 were reported the synthesis

and the conformational features of cyclic tri- tetra- hexa- octa and deca- N-benzyloxyethyl glycines

(30-34 figure 18)21a

Figure 18 Structure of cyclic tri- tetra- hexa- octa and deca- N-benzyloxyethyl glycines

It was found for the flexible eighteen-membered N-benzyloxyethyl cyclic peptoid 32 high binding

constants with the first group alkali metals (Ka ~ 106 for Na+ Li

+ and K

+) while for the rigid cisndash

transndashcisndashtrans cyclic tetrapeptoid 31 there was no evidence of alkali metals complexation The

conformational disorder in solution was seen as a propitious auspice for the complexation studies In

fact the stepwise addition of sodium picrate to 32 induced the formation of a new chemical species

whose concentration increased with the gradual addition of the guest The conformational equilibrium

between the free host and the sodium complex resulted in being slower than the NMR-time scale

giving with an excess of guest a remarkably simplified 1H NMR spectrum reflecting the formation of

a 6-fold symmetric species (Figure 19)

Figure 19 Picture of the predicted lowest energy conformation for the complex 32 with sodium

A conformational search on 32 as a sodium complex suggested the presence of an S6-symmetry axis

passing through the intracavity sodium cation (Figure 19) The electrostatic (ionndashdipole) forces stabilize

17

this conformation hampering the ring inversion up to 425 K The complexity of the rt 1H NMR

spectrum recorded for the cyclic 33 demonstrated the slow exchange of multiple conformations on the

NMR time scale Stepwise addition of sodium picrate to 33 induced the formation of a complex with a

remarkably simplified 1H NMR spectrum With an excess of guest we observed the formation of an 8-

fold symmetric species (Figure 110) was observed

Figure 110 Picture of the predicted lowest energy conformations for 33 without sodium cations

Differently from the twenty-four-membered 33 the N-benzyloxyethyl cyclic homologue 34 did not

yield any ordered conformation in the presence of cationic guests The association constants (Ka) for the

complexation of 32 33 and 34 to the first group alkali metals and ammonium were evaluated in H2Ondash

CHCl3 following Cramlsquos method (Table 11) 41

The results presented in Table 11 show a good degree

of selectivity for the smaller cations

Table 11 R Ka and G for cyclic peptoid hosts 32 33 and 34 complexing picrate salt guests in CHCl3 at 25

C figures within plusmn10 in multiple experiments guesthost stoichiometry for extractions was assumed as 11

41 K E Koenig G M Lein P Stuckler T Kaneda and D J Cram J Am Chem Soc 1979 101 3553

18

The ability of cyclic peptoids to extract cations from bulk water to an organic phase prompted us to

verify their transport properties across a phospholipid membrane

The two processes were clearly correlated although the latter is more complex implying after

complexation and diffusion across the membrane a decomplexation step42-43

In the presence of NaCl as

added salt only compound 32 showed ionophoric activity while the other cyclopeptoids are almost

inactive Cyclic peptoids have different cation binding preferences and consequently they may exert

selective cation transport These results are the first indication that cyclic peptoids can represent new

motifs on which to base artificial ionophoric antibiotics

145 Catalytic Peptoids

An interesting example of the imaginative use of reactive heterocycles in the peptoid field can be

found in the ―foldamers mimics ―Foldamers mimics are synthetic oligomers displaying

conformational ordering Peptoids have never been explored as platform for asymmetric catalysis

Kirshenbaum

reported the synthesis of a library of helical ―peptoid oligomers enabling the oxidative

kinetic resolution (OKR) of 1-phenylethanol induced by the catalyst TEMPO (2266-

tetramethylpiperidine-1-oxyl) (figure 114)44

Figure 114 Oxidative kinetic resolution of enantiomeric phenylethanols 35 and 36

The TEMPO residue was covalently integrated in properly designed chiral peptoid backbones which

were used as asymmetric components in the oxidative resolution

The study demonstrated that the enantioselectivity of the catalytic peptoids (built using the chiral (S)-

and (R)-phenylethyl amines) depended on three factors 1) the handedness of the asymmetric

environment derived from the helical scaffold 2) the position of the catalytic centre along the peptoid

backbone and 3) the degree of conformational ordering of the peptoid scaffold The highest activity in

the OKR (ee gt 99) was observed for the catalytic peptoids with the TEMPO group linked at the N-

terminus as evidenced in the peptoid backbones 39 (39 is also mentioned in figure 114) and 40

(reported in figure 115) These results revealed that the selectivity of the OKR was governed by the

global structure of the catalyst and not solely from the local chirality at sites neighboring the catalytic

centre

42 R Ditchfield J Chem Phys 1972 56 5688 43 K Wolinski J F Hinton and P Pulay J Am Chem Soc 1990 112 8251 44 G Maayan M D Ward and K Kirshenbaum Proc Natl Acad Sci USA 2009 106 13679

19

Figure 115 Catalytic biomimetic oligomers 39 and 40

146 PNA and Peptoids Tagged With Nucleobases

Nature has selected nucleic acids for storage (DNA primarily) and transfer of genetic information

(RNA) in living cells whereas proteins fulfill the role of carrying out the instructions stored in the genes

in the form of enzymes in metabolism and structural scaffolds of the cells However no examples of

protein as carriers of genetic information have yet been identified

Self-recognition by nucleic acids is a fundamental process of life Although in nature proteins are

not carriers of genetic information pseudo peptides bearing nucleobases denominate ―peptide nucleic

acids (PNA 41 figure 116)4 can mimic the biological functions of DNA and RNA (42 and 43 figure

116)

Figure 116 Chemical structure of PNA (19) DNA (20) RNA (21) B = nucleobase

The development of the aminoethylglycine polyamide (peptide) backbone oligomer with pendant

nucleobases linked to the glycine nitrogen via an acetyl bridge now often referred to PNA was inspired

by triple helix targeting of duplex DNA in an effort to combine the recognition power of nucleobases

with the versatility and chemical flexibility of peptide chemistry4 PNAs were extremely good structural

mimics of nucleic acids with a range of interesting properties

DNA recognition

Drug discovery

20

1 RNA targeting

2 DNA targeting

3 Protein targeting

4 Cellular delivery

5 Pharmacology

Nucleic acid detection and analysis

Nanotechnology

Pre-RNA world

The very simple PNA platform has inspired many chemists to explore analogs and derivatives in

order to understand andor improve the properties of this class DNA mimics As the PNA backbone is

more flexible (has more degrees of freedom) than the phosphodiester ribose backbone one could hope

that adequate restriction of flexibility would yield higher affinity PNA derivates

The success of PNAs made it clear that oligonucleotide analogues could be obtained with drastic

changes from the natural model provided that some important structural features were preserved

The PNA scaffold has served as a model for the design of new compounds able to perform DNA

recognition One important aspect of this type of research is that the design of new molecules and the

study of their performances are strictly interconnected inducing organic chemists to collaborate with

biologists physicians and biophysicists

An interesting property of PNAs which is useful in biological applications is their stability to both

nucleases and peptidases since the ―unnatural skeleton prevents recognition by natural enzymes

making them more persistent in biological fluids45

The PNA backbone which is composed by repeating

N-(2 aminoethyl)glycine units is constituted by six atoms for each repeating unit and by a two atom

spacer between the backbone and the nucleobase similarly to the natural DNA However the PNA

skeleton is neutral allowing the binding to complementary polyanionic DNA to occur without repulsive

electrostatic interactions which are present in the DNADNA duplex As a result the thermal stability

of the PNADNA duplexes (measured by their melting temperature) is higher than that of the natural

DNADNA double helix of the same length

In DNADNA duplexes the two strands are always in an antiparallel orientation (with the 5lsquo-end of

one strand opposed to the 3lsquo- end of the other) while PNADNA adducts can be formed in two different

orientations arbitrarily termed parallel and antiparallel (figure 117) both adducts being formed at room

temperature with the antiparallel orientation showing higher stability

Figure 117 Parallel and antiparallel orientation of the PNADNA duplexes

PNA can generate triplexes PAN-DNA-PNA the base pairing in triplexes occurs via Watson-Crick

and Hoogsteen hydrogen bonds (figure 118)

45 Demidov VA Potaman VN Frank-Kamenetskii M D Egholm M Buchardt O Sonnichsen S H Nielsen

PE Biochem Pharmscol 1994 48 1310

21

Figure 118 Hydrogen bonding in triplex PNA2DNA C+GC (a) and TAT (b)

In the case of triplex formation the stability of these type of structures is very high if the target

sequence is present in a long dsDNA tract the PNA can displace the opposite strand by opening the

double helix in order to form a triplex with the other thus inducing the formation of a structure defined

as ―P-loop in a process which has been defined as ―strand invasion (figure 119)46

Figure 119 Mechanism of strand invasion of double stranded DNA by triplex formation

However despite the excellent attributes PNA has two serious limitations low water solubility47

and

poor cellular uptake48

Many modifications of the basic PNA structure have been proposed in order to improve their

performances in term of affinity and specificity towards complementary oligonucleotide sequences A

modification introduced in the PNA structure can improve its properties generally in three different

ways

i) Improving DNA binding affinity

ii) Improving sequence specificity in particular for directional preference (antiparallel vs parallel)

and mismatch recognition

46 Egholm M Buchardt O Nielsen PE Berg RH J Am Chem Soc 1992 1141895 47 (a) U Koppelhus and P E Nielsen Adv Drug Delivery Rev 2003 55 267 (b) P Wittung J Kajanus K

Edwards P E Nielsen B Nordeacuten and B G Malmstrom FEBS Lett 1995 365 27 48 (a) E A Englund D H Appella Angew Chem Int Ed 2007 46 1414 (b) A Dragulescu-Andrasi S

Rapireddy G He B Bhattacharya J J Hyldig-Nielsen G Zon and D H Ly J Am Chem Soc 2006 128

16104 (c) P E Nielsen Q Rev Biophys 2006 39 1 (d) A Abibi E Protozanova V V Demidov and M D

Frank-Kamenetskii Biophys J 2004 86 3070

22

iii) Improving bioavailability (cell internalization pharmacokinetics etc)

Structure activity relationships showed that the original design containing a 6-atom repeating unit

and a 2-atom spacer between backbone and the nucleobase was optimal for DNA recognition

Introduction of different functional groups with different chargespolarityflexibility have been

described and are extensively reviewed in several papers495051

These studies showed that a ―constrained

flexibility was necessary to have good DNA binding (figure 120)

Figure 120 Strategies for inducing preorganization in the PNA monomers59

The first example of ―peptoid nucleic acid was reported by Almarsson and Zuckermann52

The shift

of the amide carbonyl groups away from the nucleobase (towards thebackbone) and their replacement

with methylenes resulted in a nucleosidated peptoid skeleton (44 figure 121) Theoretical calculations

showed that the modification of the backbone had the effect of abolishing the ―strong hydrogen bond

between the side chain carbonyl oxygen (α to the methylene carrying the base) and the backbone amide

of the next residue which was supposed to be present on the PNA and considered essential for the

DNA hybridization

Figure 121 Peptoid nucleic acid

49 a) Kumar V A Eur J Org Chem 2002 2021-2032 b) Corradini R Sforza S Tedeschi T Marchelli R

Seminar in Organic Synthesis Societagrave Chimica Italiana 2003 41-70 50 Sforza S Haaima G Marchelli R Nielsen PE Eur J Org Chem 1999 197-204 51 Sforza S Galaverna G Dossena A Corradini R Marchelli R Chirality 2002 14 591-598 52 O Almarsson T C Bruice J Kerr and R N Zuckermann Proc Natl Acad Sci USA 1993 90 7518

23

Another interesting report demonstrating that the peptoid backbone is compatible with

hybridization came from the Eschenmoser laboratory in 200753

This finding was part of an exploratory

work on the pairing properties of triazine heterocycles (as recognition elements) linked to peptide and

peptoid oligomeric systems In particular when the backbone of the oligomers was constituted by

condensation of iminodiacetic acid (45 and 46 Figure 122) the hybridization experiments conducted

with oligomer 45 and d(T)12

showed a Tm

= 227 degC

Figure 122 Triazine-tagged oligomeric sequences derived from an iminodiacetic acid peptoid backbone

This interesting result apart from the implications in the field of prebiotic chemistry suggested the

preparation of a similar peptoid oligomers (made by iminodiacetic acid) incorporating the classic

nucleobase thymine (47 and 48 figure 123)54

Figure 123 Thymine-tagged oligomeric sequences derived from an iminodiacetic acid backbone

The peptoid oligomers 47 and 48 showed thymine residues separated by the backbone by the same

number of bonds found in nucleic acids (figure 124 bolded black bonds) In addition the spacing

between the recognition units on the peptoid framework was similar to that present in the DNA (bolded

grey bonds)

Figure 124 Backbone thymines positioning in the peptoid oligomer (47) and in the A-type DNA

53 G K Mittapalli R R Kondireddi H Xiong O Munoz B Han F De Riccardis R Krishnamurthy and A

Eschenmoser Angew Chem Int Ed 2007 46 2470 54 R Zarra D Montesarchio C Coppola G Bifulco S Di Micco I Izzo and F De Riccardis Eur J Org

Chem 2009 6113

24

However annealing experiments demonstrated that peptoid oligomers 47 and 48 do not hybridize

complementary strands of d(A)16

or poly-r(A) It was claimed that possible explanations for those results

resided in the conformational restrictions imposed by the charged oligoglycine backbone and in the high

conformational freedom of the nucleobases (separated by two methylenes from the backbone)

Small backbone variations may also have large and unpredictable effects on the nucleosidated

peptoid conformation and on the binding to nucleic acids as recently evidenced by Liu and co-

workers55

with their synthesis and incorporation (in a PNA backbone) of N-ε-aminoalkyl residues (49

Figure 125)

NH

NN

NNH

N

O O O

BBB

X n

X= NH2 (or other functional group)

49

O O O

Figure 125 Modification on the N- in an unaltered PNA backbone

Modification on the γ-nitrogen preserves the achiral nature of PNA and therefore causes no

stereochemistry complications synthetically

Introducing such a side chain may also bring about some of the beneficial effects observed of a

similar side chain extended from the R- or γ-C In addition the functional headgroup could also serve as

a suitable anchor point to attach various structural moieties of biophysical and biochemical interest

Furthermore given the ease in choosing the length of the peptoid side chain and the nature of the

functional headgroup the electrosteric effects of such a side chain can be examined systematically

Interestingly they found that the length of the peptoid-like side chain plays a critical role in determining

the hybridization affinity of the modified PNA In the Liu systematic study it was found that short

polar side chains (protruding from the γ-nitrogen of peptoid-based PNAs) negatively influence the

hybridization properties of modified PNAs while longer polar side chains positively modulate the

nucleic acids binding The reported data did not clarify the reason of this effect but it was speculated

that factors different from electrostatic interaction are at play in the hybridization

15 Peptoid synthesis

The relative ease of peptoid synthesis has enabled their study for a broad range of applications

Peptoids are routinely synthesized on linker-derivatized solid supports using the monomeric or

submonomer synthesis method Monomeric method was developed by Merrifield2 and its synthetic

procedures commonly used for peptides mainly are based on solid phase methodologies (eg scheme

11)

The most common strategies used in peptide synthesis involve the Boc and the Fmoc protecting

groups

55 X-W Lu Y Zeng and C-F Liu Org Lett 2009 11 2329

25

Cl HON

R

O Fmoc

ON

R

O FmocPyperidine 20 in DMF

O

HN

R

O

HATU or PyBOP

repeat Scheme 11 monomer synthesis of peptoids

Peptoids can be constructed by coupling N-substituted glycines using standard α-peptide synthesis

methods but this requires the synthesis of individual monomers4 this is based by a two-step monomer

addition cycle First a protected monomer unit is coupled to a terminus of the resin-bound growing

chain and then the protecting group is removed to regenerate the active terminus Each side chain

requires a separate Nα-protected monomer

Peptoid oligomers can be thought of as condensation homopolymers of N-substituted glycine There

are several advantages to this method but the extensive synthetic effort required to prepare a suitable set

of chemically diverse monomers is a significant disadvantage of this approach Additionally the

secondary N-terminal amine in peptoid oligomers is more sterically hindered than primary amine of an

amino acid for this reason coupling reactions are slower

Sub-monomeric method instead was developed by Zuckermann et al (Scheme 12)56

Cl

HOBr

O

OBr

OR-NH2

O

HN

R

O

DIC

repeat Scheme 12 Sub-monomeric synthesis of peptoids

Sub-monomeric method consists in the construction of peptoid monomer from C- to N-terminus

using NN-diisopropylcarbodiimide (DIC)-mediated acylation with bromoacetic acid followed by

amination with a primary amine This two-step sequence is repeated iteratively to obtain the desired

oligomer Thereafter the oligomer is cleaved using trifluoroacetic acid (TFA) or by

hexafluorisopropanol scheme 12 Interestingly no protecting groups are necessary for this procedure

The availability of a wide variety of primary amines facilitates the preparation of chemically and

structurally divergent peptoids

16 Synthesis of PNA monomers and oligomers

The first step for the synthesis of PNA is the building of PNAlsquos monomer The monomeric unit is

constituted by an N-(2-aminoethyl)glycine protected at the terminal amino group which is essentially a

pseudopeptide with a reduced amide bond The monomeric unit can be synthesized following several

methods and synthetic routes but the key steps is the coupling of a modified nucleobase with the

secondary amino group of the backbone by using standard peptide coupling reagents (NN-

dicyclohexylcarbodiimide DCC in the presence of 1-hydroxybenzotriazole HOBt) Temporary

masking the carboxylic group as alkyl or allyl ester is also necessary during the coupling reactions The

56 R N Zuckermann J M Kerr B H Kent and W H Moos J Am Chem Soc 1992 114 10646

26

protected monomer is then selectively deprotected at the carboxyl group to produce the monomer ready

for oligomerization The choice of the protecting groups on the amino group and on the nucleobases

depends on the strategy used for the oligomers synthesis The similarity of the PNA monomers with the

amino acids allows the synthesis of the PNA oligomer with the same synthetic procedures commonly

used for peptides mainly based on solid phase methodologies The most common strategies used in

peptide synthesis involve the Boc and the Fmoc protecting groups Some ―tactics on the other hand

are necessary in order to circumvent particularly difficult steps during the synthesis (ie difficult

sequences side reactions epimerization etc) In scheme 13 a general scheme for the synthesis of PNA

oligomers on solid-phase is described

NH

NOH

OO

NH2

First monomer loading

NH

NNH

OO

Deprotection

H2NN

NH

OO

NH

NOH

OO

CouplingNH

NNH

OO

NH

N

OO

Repeat deprotection and coupling

First cleavage

NH2

HNH

N

OO

B

nPNA

B-PGs B-PGs

B-PGsB-PGs

PGt PGt

PGt

PGs Semi-permanent protecting groupPGt Temporary protecting group

Scheme 13 Typical scheme for solid phase PNA synthesis

The elongation takes place by deprotecting the N-terminus of the anchored monomer and by

coupling the following N-protected monomer Coupling reactions are carried out with HBTU or better

its 7-aza analogue HATU57

which gives rise to yields above 99 Exocyclic amino groups present on

cytosine adenine and guanine may interfere with the synthesis and therefore need to be protected with

semi-permanent groups orthogonal to the main N-terminal protecting group

In the Boc strategy the amino groups on nucleobases are protected as benzyloxycarbonyl derivatives

(Cbz) and actually this protecting group combination is often referred to as the BocCbz strategy The

Boc group is deprotected with trifluoroacetic acid (TFA) and the final cleavage of PNA from the resin

with simultaneous deprotection of exocyclic amino groups in the nucleobases is carried out with HF or

with a mixture of trifluoroacetic and trifluoromethanesulphonic acids (TFATFMSA) In the Fmoc

strategy the Fmoc protecting group is cleaved under mild basic conditions with piperidine and is

57 Nielsen P E Egholm M Berg R H Buchardt O Anti-Cancer Drug Des 1993 8 53

27

therefore compatible with resin linkers such as MBHA-Rink amide or chlorotrityl groups which can be

cleaved under less acidic conditions (TFA) or hexafluoisopropanol Commercial available Fmoc

monomers are currently protected on nucleobases with the benzhydryloxycarbonyl (Bhoc) groups also

easily removed by TFA Both strategies with the right set of protecting group and the proper cleavage

condition allow an optimal synthesis of different type of classic PNA or modified PNA

17 Aims of the work

The objective of this research is to gain new insights in the use of peptoids as tools for structural

studies and biological applications Five are the themes developed in the present thesis

1 Carboxyalkyl Peptoid PNAs Nγ-carboxyalkyl modified peptide nucleic acids (PNAs)

containing the four canonical nucleobases were prepared via solid-phase oligomerization The inserted

modified peptoid monomers (figures 126 50 and 51) were constructed through simple synthetic

procedures utilizing proper glycidol and iodoalkyl electrophiles

Figure 126 Modified peptoid monomers

Synthesis of PNA oligomers was realized by inserting modified peptoid monomers into a canonical

PNA by this way four different modified PNA oligomers were obtained (figure 127)

Figure 127 Modified PNA

Thermal denaturation studies performed in collaboration with Prof R Corradini from the University

of Parma with complementary antiparallel DNA strands demonstrated that the length of the Nγ-side

chain strongly influences the modified PNAs hybridization properties Moreover multiple negative

GTAGAT50CACTndashGlyndashNH2 52

G T50AGAT50CAC T50ndashGlyndashNH2 53

NH

NN

N

O

Base

OOO

NH

N

BaseO

O

N

NH

O

HO50

NH

NN

N

O

Base

OOO

NH

N

BaseO

O

N

NH

O

HO 51

FmocN

NOH

N

NH

O

t-BuO

O

n 50 n = 151 n = 5

28

charges on the oligoamide backbone when present on γ-nitrogen C6 side chains proved to be beneficial

for the oligomers water solubility and DNA hybridization specificity

2 Structural analysis of cyclopeptoids and their complexes The aim of this work was the

studies of structural properties of cyclopeptoids in their free and complexed form (figure 127 56 57

and 58)

N

OO

O

N

O

N

O

56

N

NN

OO

O

N

O

57

N

O

N

O ON

N

O

O58

Figure 127 N-Benzyl-cyclohexapeptoid 56 N-benzyl-cyclotetrapeptoid 57 and N-metoxyethyl-

cyclohexapeptoid 58

The synthesis of hexa- and tetra- N-benzyl glycine linear oligomers and of hexa- N-metoxyethyl

glycine linear oligomer (59 60 and 61 figure 128) was accomplished on solid-phase (2-chlorotrityl

resin) using the ―sub-monomer approach58

HON

H

O

HON

H

O

n=661n=659n=460

n n

Figure 128 linear N-Benzyl-hexapeptoid 59 linear N-benzyl tetrapeptoid 60 and linear N-

metoxyethyl-hexapeptoid 61

58

R N Zuckermann J M Kerr B H Kent and W H Moos J Am Chem Soc 1992 114 10646

29

All cycles obtained were crystallized and caractherizated by X-ray analysis in collaboration with

Dott Consiglia Tedesco from the University of Salerno and Dott Loredana Erra from European

Synchrotron Radiation Facility (ESRF) Grenoble France

3 Cationic cyclopeptoids as potential macrocyclic nonviral vectors

The aim of this work was the synthesis of three different cationic cyclopeptoids (figure 128 62 63

and 64) to assess their efficiency in DNA cell transfection in collaboration with Prof G Donofrio of

the University of Parma

N

NN

N

NNO

O

OO

O

H3N

2X CF3COO-

N

NN

N

O

H3N

NH3

H3N

4X CF3COO-

62 63

N

NN

N

NNO

O

OO

O

H3N

NH3

H3N

NH3

6X CF3COO-

64

Figure 129 Di-cationic cyclohexapeptoid 62 Tetra-cationic cyclohexapeptoid 63 Hexa-cationic

cyclohexapeptoid 64

4 Complexation with Gd3+

of carboxyethyl cyclopeptoids as possible contrast agents in

MRI Three cyclopeptoids 65 66 and 67 (figure 130) containing polar side chains were synthesized

and in collaboration with Prof S Aime of the University of Torino the complexation properties with

Gd3+

were evaluated

30

NN

OOO

OH

O

HO O

HO

O

OHO

N NN

O OO OMe

NNNO

OO

MeO

NN

OOO

OH

O

OH

O

HO O

HO

O

HO

O

OHO

NN

OOO

O

OH

O

HO

O

OHO

6566

67 Figure 129 Hexacarboxyethyl cyclohexapeptiod 65 Tricarboxyethyl ciclohexapeprtoid 66 and

tetracarboxyethyl cyclopeptoids 67

5 Cyclopeptoids as mimetic of natural defensins59

In this work some linear and

cyclopeptoids with specific side chains (-SH groups) were synthetized The aim was to introduce by

means of sulfur bridges peptoid backbone constrictions and to mimic natural defensins (figure 130

block I 68 hexa-linear and related cycles 69 and 70 block II 71 octa-linear and related cycles 72 and

73 block III 74 dodeca-linear and related cycles 75 76 and 77 block IV 78 dodeca-linear diprolinate

and related cycles 79 80 and 81)

HO

NN

NNH

OO

O

STr STr

NHBoc NHBoc68

N

NN

N

NNO

O

OO

O

NHBoc

SH

BocHN

HS

69N

NN

N

NNO

O

OO

O

NHBoc

S

BocHN

S

70

Figure 130 block I Structures of the hexameric linear (68) and corresponding cyclic 69 and 70

59

a) W Wang SM Owen D L Rudolph A M Cole T Hong A J Waring R B Lal and R I

Lehrer The Journal of Immunology 2010 515-520 b) D Yang A Biragyn D M Hoover J

Lubkowski J J Oppenheim Annu Rev Immunol 2004 181-215

31

N

NN

N

O O

O

OOO

O

SH

HS

N

NN

N

O O

O

OO

O

S

72 73

NN

OO

O

STr

71

N

O

NH

STr

HO

Figure 130 block II Structures of octameric linear (71) and corresponding cyclic 72 and 73

OHNN

N

NN

OO

O

OO

O

TrS

NOO

N

NN

NNH

OO

O

TrS

74N

N

N N

NN

N

NO

O

O O O

OOO

O

NO

SH

HS

N

N N

NN

N

NO

O

O O O

OOO

O

NO

S

75

76

OH

NN

N

OO

O

S

NO

O

N

NH

O

S

77

Figure 130 block III Structures of linear (74) and corresponding cyclic 75 76 and 77

32

HO

NN

OO O

OO

OTrS

78

NOO

N

HN

O

O STr

N

N N

NN

N

NO

O

O O O

OOO

O

NO

HS

79

SH

N

N N

NN

N

NO

O

O O O

OOO

O

NO

S

80

S

HO

NN

OO O

OO

O

S

NOO

N

HN

O

OS

81

Figure 130 block IV Structures of dodecameric linear diprolinate (78) and corresponding cyclic

79 80 and 81

33

Chapter 2

2 Carboxyalkyl Peptoid PNAs Synthesis and Hybridization Properties

21 Introduction

The considerable biological stability the excellent nucleic acids binding properties and the

appreciable chemical simplicity make PNA an invaluable tool in molecular biology60

Unfortunately

despite the remarkable properties PNA has two serious limitations low water solubility61

and poor

cellular uptake62

Considerable efforts have been made to circumvent these drawbacks and a conspicuous number of

new analogs have been proposed63

including those with the γ-nitrogen modified N-(2-aminoethyl)-

glycine (aeg) units64

In a contribution by the Nielsen group65

an accurate investigation on the Nγ-

methylated PNA hybridization properties was reported In this study it was found that the formation of

PNA-DNA (or RNA) duplexes was not altered in case of a 30 Nγ-methyl nucleobase substitution

However the hybridization efficiency per N-methyl unit in a PNA decreased with the increasing of the

N-methyl content

The negative impact of the γ-N alteration reported by Nielsen did not discouraged further

investigations The potentially informational triazine-tagged oligoglycines systems66

the oligomeric

thymine-functionalized peptoids5d

the achiral Nγ-ω-aminoalkyl nucleic acids

5a constitute convincing

example of γ-nitrogen beneficial modification In particular the Liu group contribution5a

revealed an

unexpected electrosteric effect played by the Nγ-side chain length In their stringent analysis it was

demonstrated that while short ω-amino Nγ-side chains negatively influenced the modified PNAs

hybridisation properties longer ω-amino Nγ-side chains positively modulated nucleic acids binding It

was also found that suppression of the positive ω-aminoalkyl charge (ie through acetylation) caused no

reduction in the hybridization affinity suggesting that factors different from mere electrostatic

stabilizing interactions were at play in the hybrid aminopeptoid-PNADNA (RNA) duplexes67

Considering the interesting results achieved in the case of N-(2-alkylaminoethyl)-glycine units56

and

on the basis of poor hybridization properties showed by two fully peptoidic homopyrimidine oligomers

synthesized by our group5b

it was decided to explore the effects of anionic residues at the γ-nitrogen in

a PNA framework on the in vitro hybridization properties

60 (a) Nielsen P E Mol Biotechnol 2004 26 233-248 (b) Brandt O Hoheisel J D Trends Biotechnol 2004

22 617-622 (c) Ray A Nordeacuten B FASEB J 2000 14 1041-1060 61 Vernille J P Kovell L C Schneider J W Bioconjugate Chem 2004 15 1314-1321 62 (a) Koppelhus U Nielsen P E Adv Drug Delivery Rev 2003 55 267-280 (b) Wittung P Kajanus J

Edwards K Nielsen P E Nordeacuten B Malmstrom B G FEBS Lett 1995 365 27-29 63 (a) De Koning M C Petersen L Weterings J J Overhand M van der Marel G A Filippov D V

Tetrahedron 2006 62 3248ndash3258 (b) Murata A Wada T Bioorg Med Chem Lett 2006 16 2933ndash2936 (c)

Ma L-J Zhang G-L Chen S-Y Wu B You J-S Xia C-Q J Pept Sci 2005 11 812ndash817 64 (a) Lu X-W Zeng Y Liu C-F Org Lett 2009 11 2329-2332 (b) Zarra R Montesarchio D Coppola

C Bifulco G Di Micco S Izzo I De Riccardis F Eur J Org Chem 2009 6113-6120 (c) Wu Y Xu J-C

Liu J Jin Y-X Tetrahedron 2001 57 3373-3381 (d) Y Wu J-C Xu Chin Chem Lett 2000 11 771-774 65 Haaima G Rasmussen H Schmidt G Jensen D K Sandholm Kastrup J Wittung Stafshede P Nordeacuten B

Buchardt O Nielsen P E New J Chem 1999 23 833-840 66 Mittapalli G K Kondireddi R R Xiong H Munoz O Han B De Riccardis F Krishnamurthy R

Eschenmoser A Angew Chem Int Ed 2007 46 2470-2477 67 The authors suggested that longer side chains could stabilize amide Z configuration which is known to have a

stabilizing effect on the PNADNA duplex See Eriksson M Nielsen P E Nat Struct Biol 1996 3 410-413

34

The N-(carboxymethyl) and the N-(carboxypentamethylene) Nγ-residues present in the monomers 50

and 51 (figure 21) were chosen in order to evaluate possible side chains length-dependent thermal

denaturations effects and with the aim to respond to the pressing water-solubility issue which is crucial

for the specific subcellular distribution68

Figure 21 Modified peptoid PNA monomers

The synthesis of a negative charged N-(2-carboxyalkylaminoethyl)-glycine backbone (negative

charged PNA are rarely found in literature)69

was based on the idea to take advantage of the availability

of a multitude of efficient methods for the gene cellular delivery based on the interaction of carriers with

negatively charged groups Most of the nonviral gene delivery systems are in fact based on cationic

lipids70

or cationic polymers71

interacting with negative charged genetic vectors Furthermore the

neutral backbone of PNA prevents them to be recognized by proteins which interact with DNA and

PNA-DNA chimeras should be synthesized for applications such as transcription factors scavenging

(decoy)72

or activation of RNA degradation by RNase-H (as in antisense drugs)

This lack of recognition is partly due to the lack of negatively charged groups and of the

corresponding electrostatic interactions with the protein counterpart73

In the present work we report the synthesis of the bis-protected thyminylated Nγ-ω-carboxyalkyl

monomers 50 and 51 (figure 21) the solid-phase oligomerization and the base-pairing behaviour of

four oligomeric peptoid sequences 52-55 (figure 22) incorporating in various extent and in different

positions the monomers 50 and 51

68 Koppelius U Nielsen P E Adv Drug Deliv Rev 2003 55 267-280 69 (a) Efimov V A Choob M V Buryakova A A Phelan D Chakhmakhcheva O G Nucleosides

Nucleotides Nucleic Acids 2001 20 419-428 (b) Efimov V A Choob M V Buryakova A A Kalinkina A

L Chakhmakhcheva O G Nucleic Acids Res 1998 26 566-575 (c) Efimov V A Choob M V Buryakova

A A Chakhmakhcheva O G Nucleosides Nucleotides 1998 17 1671-1679 (d) Uhlmann E Will D W

Breipohl G Peyman A Langner D Knolle J OlsquoMalley G Nucleosides Nucleotides 1997 16 603-608 (e)

Peyman A Uhlmann E Wagner K Augustin S Breipohl G Will D W Schaumlfer A Wallmeier H Angew

Chem Int Ed 1996 35 2636ndash2638 70 Ledley F D Hum Gene Ther 1995 6 1129ndash1144 71 Wu G Y Wu C H J Biol Chem 1987 262 4429ndash4432 72Gambari R Borgatti M Bezzerri V Nicolis E Lampronti I Dechecchi M C Mancini I Tamanini A

Cabrini G Biochem Pharmacol 2010 80 1887-1894 73 Romanelli A Pedone C Saviano M Bianchi N Borgatti M Mischiati C Gambari R Eur J Biochem

2001 268 6066ndash6075

FmocN

NOH

N

NH

O

t-BuO

O

n 32 n = 133 n = 5

35

Figure 22 Structures of target oligomers 52-55 T represents the modified thyminylated Nγ-ω-

carboxyalkyl monomers T50 incorporates monomer 50 T51 incorporates monomer 51

The carboxy termini of the modified mixed purinepyrimidine decamer PNA sequences were linked

to a glycinamide unit T1 and T2 represent the insertion of the modified 50 and 51 Nγ-ω-carboxyalkyl

monomer units respectively

The mixed-base sequence has been chosen since it has been proposed by Nielsen and coworkers and

subsequently used by several groups as a benchmark for the evaluation of the effect of modification of

the PNA structure on PNADNA thermal stability74

22 Results and discussion

221 Chemistry

The elaboration of monomers 50 and 51 (figure 21) suitable for the Fmoc-based oligomerization

took advantage of the chemistry utilized to construct the regular PNA monomers In particular the

synthesis of the N-protected monomer 50 started with the t-Bu-glycine (82) glycidol amination5 as

shown in scheme 21 N-fluorenylmethoxycarbonyl protection of the adduct 85 and subsequent diol

oxidative cleavage gave the labile aldehyde 86 Compound 86 was subjected to reductive amination in

the presence of methylglycine to obtain the triply protected bis-carboxyalkyl ethylenediamine key

intermediate 87

The 2-(7-aza-1H-benzotriazole-1-yl)-1133-tetramethyluronium hexafluorophosphate (HATU)

promoted condensation of 87 with thymine-1-acetic acid gave the expected tertiary amide 88

Careful LiOH-mediated hydrolysis allowed to preserve the base-labile Fmoc group affording the

target monomer unit 50

74 (a) Sforza S Tedeschi T Corradini R Marchelli R Eur J Org Chem 2007 5879ndash5885 (b) Englund E

A Appella D H Org Lett 2005 7 3465-3467 (c) Sforza S Corradini R Ghirardi S Dossena A

Marchelli R Eur J Org Chem 2000 2905-2913

36

Scheme 21 Synthesis of the PNA monomer 50 Reagents and conditions a) glycidol DMF

DIPEA 70degC 3 days 41 b) fluorenylmethoxycarbonyl chloride (Fmoc-Cl) NaHCO3 14-

dioxaneH2O overnight 63 c) NaIO4 THFH2O 2h 97 d) H2NCH2COOCH3 NaHB(AcO)3

triethylamine in CH2Cl2 overnight 70 e) thymine-1-acetic acid Et3N HATU in DMF overnight

49 f) LiOHH2O 14-dioxane H2O 0degC 30 min 69

The synthesis of compound 51 required a different strategy due to the low yields obtained in the

glycidol opening induced by the t-butyl ester of the 6-aminocaproic acid (89 see the experimental

section) A better electrophile was devised in the benzyl 2-iodoethylcarbamate (6575

Scheme 22) The

nucleophilic displacement gave the secondary amine 95 containing the Cbz-protected ethylendiamine

core Compound 95 after a straightforward protective group adjustment and a subsequent reductive

amination produced the fully protected bis-carboxyalkyl ethylenediamine key intermediate 98 This last

was reacted with thymine-1-acetic acid and benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium

hexafluorophosphate (PyBOP) as condensing agent and gave the amide 99 Finally after careful

chemoselective hydrolysis of the methyl ester the required monomer 51 was obtained in acceptable

yields

75 Bolognese A Fierro O Guarino D Longobardo L Caputo R Eur J Org Chem 2006 169-173

O

t-BuONH2 O

OH+

O

t-BuON

R

82 83 84 R = H

85 R = Fmoc

a

b

c

d

O

t-BuON

Fmoc

O

t-BuON

Fmoc

OHOH

OHN

O

e

O

t-BuON

Fmoc NO

OR

86 87

O

NH

O

88 R = CH3

50 R = H

f

37

Scheme 22 Synthesis of the PNA monomer 51 Reagents and conditions a) t-Butanol DMAP DCC CH2Cl2

overnight 58 b) H2 PdC (10 ww) acetic acid methanol 1h and 30 min quant c) Cbz-Cl CH2Cl2 0degC

overnight quant d) I2 imidazole PPh3 CH2Cl2 3h 77 e) K2CO3 CH3CN reflux overnight 67 f)

fluorenylmethoxycarbonyl chloride (Fmoc-Cl) NaHCO3 14-dioxaneH2O overnight 97 g) H2 PdC (10

ww) acetic acid methanol 1h quant h) ethyl glyoxalate NaHB(AcO)3 triethylamine in CH2Cl2 overnight

25 i) thymine-1-acetic acid Et3N PyBOP in DMF overnight 70 l) LiOH 14-dioxaneH2O 30 min 30

The oligomers 52-55 were manually assembled in a stepwise fashion on a Rink-amide NOVA-PEG

resin solid support The unmodified PNA monomers were coupled using 2-(1H-benzotriazole-1-yl)-

1133-tetramethyluronium hexafluoro-phosphate (HBTU) HATU was used for the coupling reactions

involving the less reactive secondary amino groups of the modified monomers 50 and 51 The decamers

were detached from the solid support and quantitatively deprotected from the t-butyl protecting groups

using a 91 mixture of trifluoroacetic acid and m-cresol The water-soluble oligomers were purified by

RP-HPLC yielding the desired 52-55 as pure compounds Their identity was confirmed by MALDI-

TOF mass spectrometry

222 Hybridization studies

In order to verify the ability of decamers 49-52 to bind complementary DNA UV-monitored melting

experiments were performed mixing the water-soluble oligomers with the complementary antiparallel

O

HO

NHCbz

89

5

O

t-BuO

NH2

5

a

INHCbz

9190

b

O

t-BuO

NHCbz

5

HONH2 HO

NHCbz

92 93 94

c d

e

O

t-BuO

N

5

NHR

95 R = H R = Cbz

96 R = Fmoc R = Cbzf

R

h

97 R = Fmoc R = Hg

HN

O

t-BuO

N

5

Fmoc

51 R = H

98

i

NO

OR

O

t-BuO

N

5

Fmoc

l

ON

NH

O

91 94+

99 R = CH2CH3

38

deoxyribonucleic strands (5 μM concentration ε= 260 nm) Table 21 presents the thermal stability

studies of the duplexes formed between the modified PNAs and the DNA anti-parallel strand in

comparison with the unmodified PNA

The data obtained clearly demonstrated that the distance of the negative charged carboxy group from

the oligoamide backbone strongly affects the PNADNA duplex stability In particular when the γ-

nitrogen brings an acetic acid substituent (with a single methylene distancing the oligoamide backbone

and the charged group entry 2) a drop of 54 degC in Tm of the carboxypeptoid-PNADNA(ap) duplex is

observed when compared with unmodified PNA (entry 1) Triple insertion of monomer 50 (entry 3)

results in a decrease of 26 degC per N-acetyl unit showing no Nγ-substitution detrimental additive effects

on the annealing properties In both cases the ability to discriminate closely related sequences is

magnified respect to the unmodified PNA

Table 21 Thermal stabilities (Tm degC) of modified PNADNA duplexes

Entry PNA Anti-parallel DNA

duplexa

DNA mis-matchedb

1 Ac-GTAGATCACTndashGlyndashNH2

(PNA sequence)8a

486 364

2 GTAGAT50CACTndashGlyndashNH2 (52) 432 335

3 GT50AGAT50CACT50ndashGlyndashNH2 (53) 407 344

5 G T51AGAT51CAC T51ndashGlyndashNH2 (55) 441 356

6 5lsquondashGTAGATCACTndash3lsquo

(DNA sequence)9

335 265

a5lsquondashAGTGATCTACndash3lsquo

b5lsquondashAGTGGTCTACndash3lsquo

For the binding of the Nγ-caproic acid derivatives with the full-matched antiparallel DNA the table

shows an evident increase of the affinity (entry 4 and 5) when compared with the modified sequences

with shorter side chains (entry 2 and 3) Comparison with the corresponding aegPNA showed for the

single insertion a 38 degC Tm drop while for triple substitution a Tm decrease of 15 degC per Nγ-alkylated

monomer It is also worth noting in both 54 and 55 the slight increase of the binding specificity (ΔTm =

56 degC and 08 degC entry 4 and 5) respect to unmodified PNA

In previous studies reporting the performances of backbone modified PNA containing negatively

charged monomers derived from amino acids the drop in melting temperature was found to be 33 degC in

the case of L-Asp monomer and 23deg C in the case of D-Glu The present results are in line with these

data with a decrease in melting temperatures which still allows stronger binding than natural DNA

(entry 6) Thus it is possible to introduce negatively charged groups via alkylation of the amide nitrogen

in the PNA backbone without significant loss of stability of the PNA-DNA duplex provided that a five

methylene spacer is used

39

23 Conclusions

In this work we have constructed two orthogonally protected N--carboxy alkylated units The

successful insertion in PNA-based decamers through standard solid-phase synthesis protocols and the

following hybridization studies in the presence of DNA antiparallel strand demonstrate that the N-

substitution with negative charged groups is compatible with the formation of a stable PNADNA

duplex The present study also extends the observation that correlates the efficacy of the nucleic acids

hybridization with the length of the N alkyl substitution

5a expanding the validity also to N

--negative

charged side chains The newly produced structures can create new possibilities for PNA with

functional groups enabling further improvement in their ability to perform gene-regulation

24 Experimental section

241 General Methods

All reactions involving air or moisture sensitive reagents were carried out under a dry argon or

nitrogen atmosphere using freshly distilled solvents Tetrahydrofuran (THF) was distilled from LiAlH4

under argon Toluene and CH2Cl2 were distilled from CaH2 Glassware was flame-dried (005 Torr)

prior to use When necessary compounds were dried in vacuo over P2O5 or by azeotropic removal of

water with toluene under reduced pressure Starting materials and reagents purchased from commercial

suppliers were generally used without purification unless otherwise mentioned Reaction temperatures

were measured externally reactions were monitored by TLC on Merck silica gel plates (025 mm) and

visualized by UV light I2 or by spraying with H2SO4-Ce(SO4)2 phosphomolybdic acid or ninhydrin

solutions and drying Flash chromatography was performed on Merck silica gel 60 (particle size 0040-

0063 mm) and the solvents employed were of analytical grade Yields refer to chromatographically and

spectroscopically (1H- and

13C-NMR) pure materials The NMR spectra were recorded on Bruker DRX

400 (1H at 40013 MHz

13C at 10003 MHz) Bruker DRX 250 (

1H at 25013 MHz

13C at 6289 MHz)

and Bruker DRX 300 (1H at 30010 MHz

13C at 7550 MHz) spectrometers Chemical shifts () are

reported in ppm relatively to the residual solvent peak (CHCl3 = 726 13

CDCl3 = 770 CD2HOD

= 334 13

CD3OD = 490) and the multiplicity of each signal is designated by the following

abbreviations s singlet d doublet t triplet q quartet quint quintuplet m multiplet br broad

Coupling costants (J) are quoted in Hz Homonuclear decoupling COSY-45 and DEPT experiments

completed the full assignment of each signal Elemental analyses were performed on a CHNS-O

FlashEA apparatus (Thermo Electron Corporation) and are reported in percent abundance High

resolution ESI-MS spectra were performed on a Q-Star Applied Biosystem mass spectrometer ESI-MS

analysis in positive ion mode was performed using a Finnigan LCQ Deca ion trap mass spectrometer

(ThermoFinnigan San Josegrave CA USA) and the mass spectra were acquired and processed using the

Xcalibur software provided by Thermo Finnigan Samples were dissolved in 11 CH3OHH2O 01

formic acid and infused in the ESI source by using a syringe pump the flow rate was 5 μlmin The

capillary voltage was set at 40 V the spray voltage at 5 kV and the tube lens offset at -40 V The

capillary temperature was 220 degC MALDI TOF mass spectrometric analyses were performed on a

PerSeptive Biosystems Voyager-De Pro MALDI mass spectrometer in the Linear mode using -cyano-

4-hydroxycinnamic acid as the matrix HPLC analyses were performed on a Jasco BS 997-01 series

equipped with a quaternary pumps Jasco PU-2089 Plus and an UV detector Jasco MD-2010 Plus The

40

resulting residues were purified by semipreparative reverse-phase C18 (Waters Bondapak 10 μm

125Aring 78 times 300 mm)

242 Chemistry

Tert-butyl 2-(23-dihydroxypropylamino)acetate (55)

To a solution of glycidol (83 436 μL 656 mmol) in DMF (5 mL) glycine t-butyl ester (82 100 g

596 mmol) in DMF (10 mL) and DIPEA (1600 μL 894 mmol) were added The reaction mixture was

refluxed for three days NaHCO3 (050 g 596 mmol) was added and the solvent was concentrated in

vacuo to give the crude product which was purified by flash chromatography (CH2Cl2CH3OHNH3 20

M solution in ethyl alcohol from 100001 to 881201) to give 84 (050 g 41) as a yellow pale oil

[Found C 527 H 94 C9H19NO4 requires C 5267 H 933] Rf (97301 CH2Cl2CH3OHNH3

20M solution in ethyl alcohol) 036 H (40013 MHz CDC13) 142 (9H s (CH3)3C) 262 (1H dd J

120 77 Hz CHHCH(OH)CH2OH) 271 (1H dd J 120 29 Hz CHHCH(OH)CH2OH) 328 (2H br

s CH2COOt-Bu) 351 (1H dd J 110 54 Hz CH2CH(OH)CHHOH) 362 (1H dd J 110 12 Hz

CH2CH(OH)CHHOH) 372 (1H m CH2CH(OH)CH2OH) C (10003 MHz CDCl3) 292 526 531

664 716 827 1728 mz (ES) 206 (MH+) (HRES) MH

+ found 2061390 C9H20NO4

+ requires

2061392

(9H-fluoren-9-yl) methyl (tert-butoxycarbonyl) methyl 23dihydroxypropylcarbamate (85)

To a solution of 84 (0681 g 333 mmol) in a 11 mixture of 14-dioxanewater (46 mL) NaHCO3

(0559 g 666 mmol) was added The mixture was sonicated until complete dissolution and Fmoc-Cl

(103 g 399 mmol) was added The reaction mixture was stirred overnight then through addition of a

saturated solution of NaHSO4 the pH was adjusted to 3 and the solvent was concentrated in vacuo to

remove the excess of 14-dioxane The water layer was extracted with CH2Cl2 (three times) the organic

phase was dried over MgSO4 filtered and the solvent evaporated in vacuo to give the crude product

which was purified by flash chromatography (CH2Cl2CH3OH from 1000 to 9010) to give 85 (090 g

63) as a yellow pale oil [Found C 674 H 69 C24H29NO6 requires C 6743 H 684] Rf

(95501 CH2Cl2CH3OHNH3 20M solution in ethyl alcohol) 044 H (30010 MHz CDC13 mixture

of rotamers) 145 (9H s (CH3)3C) 304-325 (17 H m CH2CH(OH)CH2OH) 340 (03 H m

CH2CH(OH)CH2OH) 343-392 (3H m CH2CH(OH)CH2OH CH2CH(OH)CH2OH) 393 (2H br s

CH2COOt-Bu) 422 (09H m CH-Fmoc and CH2-Fmoc) 442 (14H br d J 90 Hz CH2-Fmoc) 461

(07H m J 90 Hz CH-Fmoc) 729 (2H br t J 70 Hz Ar (Fmoc)) 738 (2H br t J 70 Hz Ar

(Fmoc)) 757 (2H br d J 90 Hz Ar (Fmoc)) 776 (2H br d J 90 Hz Ar (Fmoc) C (7550 MHz

CDCl3 mixture of rotamers) 282 474 521 523 529 532 635 640 673 681 685 701 705

831 1201 1202 1249 1252 1273 1279 1280 1415 1438 1564 1573 1704 1713 mz (ES)

428 (MH+) (HRES) MH

+ found 4282070 C24H30NO6

+ requires 4282073

(9H-fluoren-9-yl) methyl (tert-butoxycarbonyl) methylformylmethylcarbamate (86)

To a solution of 85 (080 g 187 mmol) in a 51 mixture of THF and water (5 mL) sodium periodate

(044 g 206 mmol) was added in one portion The mixture was sonicated for 15 min and stirred for

another 2 hours at room temperature The reaction mixture was filtered the filtrate was washed with

CH2Cl2 and the solvent evaporated in vacuo The crude product was dissolved in CH2Cl2H2O and the

organic phase was dried over MgSO4 filtered and the solvent evaporated in vacuo to give the labile

41

aldehyde 86 (072 g 97) as white solid Rf (928 CH2Cl2CH3OH) 056 crude 86 was used

immediately in the subsequent reductive amination reaction H (30010 MHz CDC13 mixture of

rotamers) 143 (405H s (CH3)3C) 145 (495H s (CH3)3C) 381 (09H br s CH2COO-tBu) 399 (2H

br s CH2CHO) and CH2COO-tBu overlapped) 408 (11H s CH2CHO) 422-419 (1H m CH-

Fmoc) 442 (11H d J 60 Hz CH2-Fmoc) 450 (09H d J 60 Hz CH2-Fmoc) 729 (09H br t J 70

Hz Ar (Fmoc)) 738 (11H t J 70 Hz Ar (Fmoc)) 749 (09H d J 90 Hz Ar (Fmoc)) 756 (11H

d J 90 Hz Ar (Fmoc)) 773 (2H m Ar (Fmoc)) 935 (045H br s CHO) 964 (055H br s CHO)

C (7550 MHz CDCl3) 282 473 506 508 578 584 681 686 826 827 1202 1249 1252

1273 1280 1415 1438 1439 1560 1564 1686 1687 1987 mz (ES) 396 (MH+) (HRES) MH

+

found 3961809 C23H26NO5+ requires 3961811

(9H-fluoren-9-yl) methyl (tert-butoxycarbonyl) methyl 2-

((methoxycarbonyl)methylamino)ethylcarbamate (87)

To a solution of crude aldehyde 86 (072 g 183 mmol) in dry CH2Cl2 (12 mL) a solution of glycine

methyl ester hydrochloride (030 g 239 mmol) and Et3N (041 mL 293 mmol) was added The

reaction mixture was stirred for 1 h Sodium triacetoxyborohydride (078 g 366 mmol) was then added

and the reaction mixture was stirred overnight at room temperature The resulting mixture was washed

with an aqueous saturated solution of NaHCO3 and the aqueous phase extracted with CH2Cl2 (three

times) The organic phase was dried over MgSO4 filtered and the solvent evaporated in vacuo to give

the crude product which was purified by flash chromatography (AcOEtpetroleum etherNH3 20M

solution in ethyl alcohol from 406001 to 901001) to give 87 (060 g 70) as a colorless oil

[Found C 667 H 69 C26H32N2O6 requires C 6665 H 688] Rf (98201 CH2Cl2CH3OHNH3

20M solution in ethyl alcohol) 063 H (40013 MHz CDC13 mixture of rotamers) 145 (9H s

(CH3)3C) 256 (09H t J 60 Hz N(Fmoc)CH2CH2NH) 283 (11H t J 60 N(Fmoc)CH2CH2NH)

327 (09H t J 60 Hz N(Fmoc)CH2CH2NH) 329 (09H s CH2COOMe) 344 (11H s

CH2COOMe) 349 (11H t J 60 Hz N(Fmoc)CH2CH2NH) 372 (3H s CH3) 391 (09H s

CH2COOtBu) 396 (11H s CH2COOtBu) 421 (045H t J 60 Hz CH2CHFmoc) 426 (055H t J

60 Hz CH2CHFmoc) 437 (11H d J 60 Hz CH2CHFmoc) 451 (09H d J 60 Hz CH2CHFmoc)

729 (2 H t J 70 Hz Ar (Fmoc)) 739 (2 H t J 70 Hz Ar (Fmoc)) 758 (2 H m Ar (Fmoc)) 775

(2 H d J 70 Hz Ar (Fmoc)) C (10003 MHz CDCl3) 278 470 472 474 482 486 501 503

505 533 672 676 815 817 1197 1247 1249 1268 1275 1410 1437 1560 1562 1687

1694 1719 1721 mz (ES) 469 (MH+) (HRES) MH

+ found 4692341 C26H33N2O6

+ requires

4692339

Compound 88

To a solution of 87 (060 g 128 mmol) in DMF (30 mL) thymine-1-acetic acid (035 g 190 mmol)

HATU (073 g 190 mmol) and triethylamine (054 mL 384 mmol) were added The reaction mixture

was stirred overnight concentrated in vacuo dissolved in CH2Cl2 (20 mL) and washed 1M HCl

solution The aqueous layer was extracted with CH2Cl2 (three times) The combined organic phases

were dried over MgSO4 filtered and the solvent evaporated in vacuo to give a crude material which

was purified by flash chromatography (AcOEtpetroleum ether from 3070 to 1000) to give 88 (040 g

49) as yellow oil [Found C 644 H 62 C34H39N3O9 requires C 6444 H 620] Rf (82

42

AcOEtpetroleum ether) 038 H (30010 MHz CDC13 mixture of rotamers) 139-144 (9H m

(CH3)3C) 188 (3H br s CH3-thymine) 299 (03H m CH2CH2N(Fmoc)) 308 (03H m

CH2CH2N(Fmoc)) 352 (28H m CH2CH2N(Fmoc) and CH2CH2N(Fmoc)) 377-389 (36H m

CH2CH2N(Fmoc) and CH3OOC) 396-438 (6H m CH2-thymine CH2COOCH3 CH2COO-tBu) 445-

480 (3H m CH(Fmoc) and CH2CH(Fmoc)) 690-706 (1H complex signal CH-thymine) 728 (2H

m Ar (Fmoc)) 741 (2H t J 70 Hz Ar (Fmoc)) 755 (2 H d J 70 Hz Ar (Fmoc)) 775 (2H t J 70

Hz Ar (Fmoc)) 886 (1H br s NH-thymine) C (755 MHz CDCl3) 125 282 316 366 472 474

475 478 482 491 506 518 521 525 530 665 682 823 825 1105 1202 1245 1248

12514 1253 1273 1274 1275 1280 1281 1413 1414 1438 1440 1511 1564 1627 1644

1691 1692 mz (ES) 634 (MH+) (HRES) MH

+ found 6342767 C34H40N4O9

+ requires 6342765

Compound 50

To a solution of 88 (040 g 063 mmol) in a 11 mixture of 14-dioxanewater (8 mL) at 0 degC

LiOHH2O (58 mg 139 mmol) was added The reaction mixture was stirred for 30 minutes and a

saturated solution of NaHSO4 was added until pH~3 The aqueous layer was extracted with CH2Cl2

(three times) and once with AcOEt The combined organic phases were dried over MgSO4 filtered and

the solvent evaporated in vacuo to give a crude material which was purified by flash chromatography

(CH2Cl2CH3OH AcOH from 95501 to 802001) to give 50 (027 g 69) as a white solid [Found

C 630 H 60 C33H37N3O9 requires C 6396 H 602] Rf (9101 CH2Cl2CH3OHNH3 20M

solution in ethyl alcohol) 012 H (25013 MHz CDC13 mixture of rotamers) 139-143 (9H m

(CH3)3C) 183 (3H br s CH3 (thymine)) 303 (03H m CH2CH2N(Fmoc)) 317 (03H m

CH2CH2N(Fmoc)) 355-372 (28H m CH2CH2N(Fmoc) and CH2CH2N(Fmoc)) 395-406 (66H m

CH2CH2N(Fmoc) CH2-thymine CH2COOH CH2COO-tBu) 414-477 (3H m CH(Fmoc) and

CH2CH(Fmoc)) 697-711 (1H complex signal CH-thymine) 723-740 (4H m Ar (Fmoc)) 755 (2

H d J 70 Hz Ar (Fmoc)) 775 (2H m Ar (Fmoc)) 1000 (1H br s NH-thymine) C (7550 MHz

CDCl3) 123 282 299 464 473 487 502 509 536 683 823 826 1108 1202 1249 1252

1253 1273 1275 1280 1414 1421 1438 1440 1517 1566 1650 1652 1682 1690 1692

1723 mz (ES) 620 (MH+) (HRES) MH

+ found 6202611 C33H38N3O9

+ requires 6202608

Benzyl 5-(tert-butoxycarbonyl)pentylcarbamate (90)

To a solution of 89 (300 g 113 mmol) DMAP (014 g 113 mmol) and t-BuOH (130 mL 139

mmol) in dry CH2Cl2 (50 mL) a solution of DCC (136 mL 136 mmol 10 M in CH2Cl2) was added

The reaction mixture was filtered the filtrate washed with CH2Cl2 and the solvent evaporated in vacuo

to give the crude product which was purified by flash chromatography (AcOEtpetroleum ether from

1090 to 1000) to give 90 (210 g 58) as white solid [Found C 672 H 84 C14H19NO4 requires C

6726 H 847] Rf (973 CH2Cl2CH3OH) 084H (40013 MHz CDC13) 131 (2H q J 65 Hz

CH2CH2CH2COOt-Bu) 141 (9H s COOC(CH3)3) 148 (2H q J 65 Hz CH2CH2CH2NH) 156 (2H

q J 65 Hz CH2CH2CH2COOt-Bu) 218 (2H t J 65 Hz CH2CH2CH2COOt-Bu) 315 (2H q J 65

Hz CH2CH2CH2NH) 491 (1H br s NH) 507 (2H br s CH2Bn) 732 (5H m Ar) C (7550 MHz

CDCl3) 245 260 280 295 353 408 664 799 1279 1280 1283 1366 1563 1728 mz (ES)

322 (MH+) (HRES) MH

+ found 3222015 C18H28NO4

+ requires 3222018

43

Tert-butyl 6-aminohexanoate (91)

To a solution of 90 (195 g 607 mmol) in dry MeOH (150 mL) acetic acid (139 mL 240 mmol)

and palladium on charcoal (10 ww 019 g) were added The reaction mixture was stirred under a

hydrogen atmosphere at room temperature for 1 h and filtered through celite The solvent was

evaporated in vacuo to give crude 91 (113 g 100 colorless oil) which was used in the next step

without purification Rf (955 CH2Cl2CH3OH) 046 H (40013 MHz CDC13) 133 (2H q J 65 Hz

CH2CH2CH2COOt-Bu) 139 (9H s COOC(CH3)3) 155 (2H q J 65 Hz CH2CH2CH2CH2CH2NH)

162 (2H q J 65 Hz CH2CH2CH2COOt-Bu) 217 (2H t J 65 Hz CH2CH2CH2COOt-Bu) 284 (2H t

J 65 Hz CH2CH2CH2NH) C (7550 MHz CDCl3) 243 258 272 280 351 394 802 1772 mz

(ES) 188 (MH+) (HRES) MH

+ found 1881647 C10H22NO2

+ requires 1881651

Benzyl 2-hydroxyethylcarbamate (93)

To a solution of ethanolamine (92 200 g 328 mmol) in dry CH2Cl2 (30 mL) at 0degC a solution Cbz-

Cl (373 mL 262 mmol) in dry CH2Cl2 (20 mL) was slowly added The reaction mixture was stirred for

2 hours at 0degC and at room temperature overnight The resulting mixture was washed with an aqueous

saturated solution of NaHCO3 and the aqueous phase extracted with CH2Cl2 (three times) The organic

phase was dried over MgSO4 filtered and the solvent evaporated in vacuo to give crude 93 (511 g

100 yellow pale oil) which was used in the next step without purification Rf (928 CH2Cl2CH3OH)

047 H (25013 MHz CDC13) 336 (2H br t J 65 Hz CH2OH) 371 (2H q J 65 Hz CH2NH) 511

(2H s CH2Bn) 735 (5H m Ar) C (6289 MHz CDCl3) 433 617 667 1279 1280 1284 1362

1570 mz (ES) 196 (MH+) (HRES) MH

+ found 1960970 C10H14NO3

+ requires 1960974

Benzyl 2-iodoethylcarbamate (94)

To a solution of PPh3 (266 g 102 mmol) in CH2Cl2 (10 mL) I2 (259 g 102 mmol) in CH2Cl2 (10

mL) was slowly added The reaction mixture was stirred for 30 min Imidazole (139 g 204 mmol) in

CH2Cl2 (10 mL) was then added and the reaction mixture was stirred for further 30 min Finally 93

(100 g 513 mmol) was added and the reaction mixture stirred for 3 hours The resulting mixture was

washed with an aqueous saturated solution of NaHCO3 and 10 ww of Na2S2O3 and the aqueous phase

extracted with CH2Cl2 (three times) The organic phase was dried over MgSO4 filtered and the solvent

evaporated in vacuo to give a crude material which was purified by flash chromatography

(AcOEtpetroleum ether from 0100 to 1000) to give 94 (120 g 77) as white amorphous solid

[Found C 394 H 40 C10H12INO2 requires C 3936 H 396] Rf (64 AcOEtpetroleum ether)

088H (25013 MHz CDC13) 325 (2H t J 65 Hz CH2I) 355 (2H q J 65 Hz CH2NH) 511 (2H

s CH2Bn) 736 (5H m Ar) C (6289 MHz CDCl3) 51 430 665 1278 1281 1282 1360 1558

mz (ES) 306 (MH+) (HRES) MH

+ found 3059989 C10H13INO2

+ requires 3059991

Benzyl 2-(5-(tert-butoxycarbonyl)pentylamino) ethylcarbamate (95)

To a solution of 91 (035 g 187 mmol) in dry acetonitrile (10 mL) at reflux K2CO3 (088 g 638

mmol) was added The reaction mixture was stirred for 10 min After that a solution of 94 (040 g 131

mmol) in dry acetonitrile (5 mL) was added and the reaction mixture was stirred at reflux overnight

The product was filtered and the crude was purified by flash column chromatograph (CH2Cl2CH3OH

from 1000 to 9010) to give 95 (032 g 67) as yellow light oil [Found C 659 H 86 C20H32N2O4

requires C 6591 H 862] Rf (937 CH2Cl2CH3OH) 071H (30010 MHz CDC13) 135 (2H q J

65 Hz CH2CH2CH2CH2CH2NH) 143 (9H s COOC(CH3)3) 157 (2H q J 60 Hz

CH2CH2CH2CH2CH2NH) 167 (2H q J 60 Hz CH2CH2CH2CH2CH2NH) 221 (2H t J 60 Hz

44

OOCCH2CH2) 282 (2H t J 60 Hz CH2CH2CH2CH2CH2NH) 299 (2H t J 60 Hz

CONHCH2CH2NH) 346 (2H q J 60 Hz CONHCH2CH2NH) 509 (2H s CH2Ar) 573 (1H br s

NHCOO) 734 (5H m Ar) C (7550 MHz CDCl3) 244 262 279 350 393 484 486 666 799

1279 1280 1283 1361 1566 1728 mz (ES) 365 (MH+) (HRES) MH

+ found 3652437

C20H33N2O4+ requires 3652440

Compound (96)

(148 mg 176 mmol) was added The mixture was sonicated until complete dissolution and Fmoc-Cl

saturated of NaHSO4 the pH was adjusted to 3 and the solvent was concentrated in vacuo to remove the

excess of dioxane The water layer was extracted with CH2Cl2 (three times) the organic phase was dried

over MgSO4 filtered and the solvent evaporated in vacuo to give the crude product which was purified

by flash chromatography (CH2Cl2CH3OH from 1000 to 982) to give 96 (050 g 97) as a yellow

light oil [Found C 717 H 73 C35H42N2O6 requires C 7165 H 722] Rf (955 CH2Cl2CH3OH)

061 H (40013 MHz CDC13 mixture of rotamers) 108-160 (15H m CH2CH2CH2CH2CH2N

COOC(CH3)3) 216 (2H t J 60 Hz CH2COO) 285 (08H br s CH2CH2CH2CH2CH2N) 296 (24H

CONHCH2CH2N and CH2CH2CH2CH2CH2N) 313 (08H br s CONHCH2CH2N) 330 (2H br s

CONHCH2CH2N) 419 (1H br s CH2CHFmoc) 453-457 (2H br s CH2CHFmoc) 505 (2H br s

CH2Ar) 729 (7H m Ar (Cbz) and Ar (Fmoc)) 738 (2H t J 70 Hz Ar (Fmoc)) 755 (2 H d J 70

Hz Ar (Fmoc)) 776 (2H t J 70 Hz Ar (Fmoc)) C (6289 MHz CDCl3) 245 259 278 279 352

392 396 462 468 471 476 663 797 1196 1244 1268 1274 1278 1282 1364 1411

1437 1556 1563 1727 mz (ES) 587 (MH+) (HRES) MH

+ found 5873120 C35H43N2O6

+ requires

5873121

Compound (97)

To a solution of 96 (150 mg 026 mmol) in dry MeOH (9 mL) acetic acid (29 μL 0512 mmol) and

palladium on charcoal (10ww 15 mg) were added The reaction mixture was stirred under a

evaporated in vacuo to give crude 97 (118 mg 100 colorless oil) which was used in the next step

without purification Rf (955 CH2Cl2CH3OH) 013 H (30010 MHz CDC13 mixture of rotamers)

105-160 (15H m CH2CH2CH2CH2CH2N COOC(CH3)3) 215 (2H t J 60 Hz CH2COO) 260 (06H

br s CH2CH2CH2CH2CH2N) 290-320 (4H CONHCH2CH2N CH2CH2CH2CH2CH2N

CONHCH2CH2N and CONHCH2CH2N) 338 (14H br s CONHCH2CH2N) 419 (1H br s

CH2CHFmoc) 452 (2H m CH2CHFmoc) 738 (4H m Ar (Fmoc)) 754 (2 H d J 70 Hz Ar

(Fmoc)) 774 (2H t J 70 Hz Ar (Fmoc)) C (10003 MHz CDCl3) 226 244 247 259 261 281

351 254 388 424 465 473 479 534 670 801 1198 1199 1240 1246 1269 1271 1277

1405 1413 1438 1490 1558 1571 1727 1729 mz (ES) 453 (MH+) (HRES) MH

+ found

4532740 C27H37N2O4+ requires 4532748

Compound (98)

To a solution of 97 (115 mg 026 mmol) in CH2Cl2 (5 mL) ethyl glyoxalate (33 μL 033 mmol)

Et3N (54 μL 038 mmol) and NaHB(OAc)3 (109 mg 052 mmol) were added The reaction mixture was

45

stirred overnight The resulting mixture was washed with an aqueous saturated solution of NaHCO3 and

the aqueous phase extracted with CH2Cl2 (three times) The organic phase was dried over MgSO4

filtered and the solvent evaporated in vacuo to give the crude product which was purified by flash

chromatography (AcOEtpetroleum ether from 6040 to 1000) to give 98 (35 mg 25) as white light

oil [Found C 692 H 79 C31H42N2O6 requires C 6912 H 786] Rf (95501

CH2Cl2CH3OHNH3 20M solution in ethyl alcohol) 046 H (30010 MHz CDC13 mixture of

rotamers) 100-180 (18H m CH2CH2CH2CH2CH2N COOC(CH3)3 and COOCH2CH3) 218 (2H t J

60 Hz CH2COOC(CH3)3) 246 (08H br s CH2CH2CH2CH2CH2N) 274 (12H br s

CH2CH2CH2CH2CH2N) 290-345 (6H m COCH2NHCH2CH2N) 418-423 (3H m COOCH2CH3

CH2CHFmoc) 452 (2H m CH2CHFmoc) 731 (2 H t J 7 Hz Ar (Fmoc)) 739 (2 H t J 7 Hz Ar

(Fmoc)) 757 (2 H d J 7 Hz Ar (Fmoc)) 775 (2H t J 7 Hz Ar (Fmoc)) C (10003 MHz CDCl3

mixture of rotamers) 141 247 261 280 353 468 473 478 506 607 665 799 1198 1246

1269 1275 1413 1440 1559 1562 1722 1729 mz (ES) 539 (MH+) (HRES) MH

+ found

5393117 C31H43N2O6+ requires 5393121

Compound (99)

To a solution of 98 (100 mg 0186 mmol) in DMF (6 mL) thymine-1-acetic acid (55 mg 030

mmol) PyBOP (154 mg 030 mmol) and triethylamine (83 μL 060 mmol) were added The reaction

mixture was stirred overnight concentrated in vacuo dissolved in CH2Cl2 (20 mL) and washed 1M HCl

was purified by flash chromatography (AcOEtpetroleum ether from 3070 to 1000) to give 99 (92

mg 70) as amorphous white solid [Found C 647 H 69 C38H48N4O9 requires C 6476 H 686]

Rf (640 AcOEtpetroleum ether) 011 H (25013 MHz CDC13 mixture of rotamers) 100-160 (18H

m CH2CH2CH2CH2CH2N COOC(CH3)3 and COOCH2CH3) 189 (3H s CH3-thymine) 218 (2H m

CH2COOC(CH3)3) 295-365 (6H m NHCH2CH2NCH2) 400-480 (9H m CH2OOCCH2NHCH2

CH2CHFmoc and CH2-thymine) 694-700 (1H complex signal CH-thymine) 739 (2 H t J 70 Hz

Ar (Fmoc)) 742 (2 H t J 70 Hz Ar (Fmoc)) 756 (2 H d J 70 Hz Ar (Fmoc)) 776 (2H t J 70

Hz Ar (Fmoc)) 843 (1H br s NH-thymine) C (7550 MHz CDCl3 mixture of rotamers) 121 139

246 260 280 353 464 467 472 478 481 507 617 669 801 1106 1110 1199 1246

1270 1276 1413 1438 1439 1512 1564 1643 1677 1689 1730 mz (ES) 705 (MH+)

(HRES) MH+ found 7053498 C38H49N4O9

+ requires 7053500

Compound (51)

To a solution of 99 (175 mg 025 mmol) in a 11 mixture of 14-dioxanewater (6 mL) at 0 degC

LiOHH2O (23 mg 055 mmol) was added The reaction mixture was stirred for 30 minutes and

saturated solution of NaHSO4 added until pH~3 The aqueous layer was extracted with CH2Cl2 (three

times) and once with AcOEt The combined organic phases were dried over MgSO4 filtered and the

solvent evaporated in vacuo to give a crude material which was purified by flash chromatography

(CH2Cl2CH3OH from 955 to 8020) to give 51 (50 mg 30) as amorphous white solid [Found C

640 H 66 C36H44N4O9 requires C 6389 H 655] Rf (9101 CH2Cl2CH3OHNH3 20M solution

in ethyl alcohol) 022 H (40013 MHz CDC13 mixture of rotamers) 100-150 (15H m

CH2CH2CH2CH2CH2N and COOC(CH3)3) 186 (3H s CH3-thymine) 217 (2H m J 60 Hz

CH2COOC(CH3)3) 290-370 (6H m NHCH2CH2NCH2) 390-485 (7H m OCCH2NHCH2

46

CH2CHFmoc and CH2-thymine) 702 (1H br s CH-thymine) 710-745 (4H m Ar (Fmoc)) 757 (2

H d J 70 Hz Ar (Fmoc)) 777 (2H t J 70 Hz Ar (Fmoc)) 1000 (1H br s NH-thymine) C

(10003 MHz CDCl3 mixture of rotamers) 135 227 259 260 273 288 294 297 366 367

458 478 485 488 496 547 683 814 816 1118 1119 1212 1259 1265 1283 1290

1295 1303 1391 1426 1429 1450 1451 1526 1577 1662 1690 1727 1744 mz (ES) 677

(MH+) (HRES) MH

+ found 6773185 C36H45N4O9

+ requires 6773187

Low yield synthesis of tert-butyl 6-(23-dihydroxypropylamino) hexanoate and unwanted

tert-butyl 6-(bis(23-dihydroxypropyl) amino) hexanoate

To a solution of glycidol (83 134 μL 202 mmol) in DMF (3 mL) t-butyl 6-aminohexanoate (91

456 mg 244 mmol) in DMF (3 mL) and DIPEA (055 mL 317 mmol) were added The reaction

mixture was refluxed for three days NaHCO3 (320 mg 179 mmol) was added and the solvent was

concentrated in vacuo to give the crude product which was purified by flash chromatography

(CH2Cl2CH3OHNH3 20M solution in ethyl alcohol from 100001 to 881201) to give 100 (60 mg

11) and 101 (320 mg 47) 100 yellow pale oil [Found C 598 H 105 C13H27NO4 requires C

5974 H 1041] Rf (901001 CH2Cl2CH3OHNH3 20M solution in ethyl alcohol) 031 H (30010

MHz CDC13) 131 (2H q J 65 Hz CH2CH2CH2COOt-Bu) 143 (9H s COOC(CH3)3) 152 (2H

quint J 65 Hz CH2CH2CH2NH) 157 (2H q J 65 Hz CH2CH2CH2COOt-Bu) 220 (2H t J 65 Hz

CH2CH2CH2COOt-Bu) 258 (2H m CH2NHCH2CH(OH)CH2(OH)) 269 (1H dd J 150 60 Hz

NHCHHCH(OH)CH2(OH)) 280 (1H dd J 150 30 Hz CHHCH(OH)CH2(OH)) 359 (1H dd J 90

30 Hz CH2CH(OH)CHH(OH)) 370 (1H dd J 90 30 Hz CH2CH(OH)CHH(OH)) 377 (1H m

CH2CH(OH)CH2(OH)) C (6289 MHz CDCl3) 246 264 279 287 352 492 518 651 697

800 1730 mz (ES) 262 (MH+) (HRES) MH

+ found 2622017 C13H28NO4

+ requires 2622018 101

yellow oil [Found C 573 H 98 C16H33NO6 requires C 5729 H 992] Rf (901001

diastereoisomers) 127 (2H q J 65 Hz CH2CH2CH2COOt-Bu) 143 (11H m COOC(CH3)3 and

CH2CH2CH2NH) 157 (2H q J 65 Hz CH2CH2CH2COOt-Bu) 219 (2H t J 65 Hz

CH2CH2CH2COOt-Bu) 240-260 (6H m CH2NH[(CH2CH(OH)CH2(OH)]2) 350 (2H m

NH[CH2CH(OH)CHH(OH)]2) 363 (2H m NH[CH2CH(OH)CHH(OH)]2) 377 (2H m

NH[CH2CH(OH)CH2(OH)]2) C (6289 MHz CDCl3 mixture of diastereoisomers) 261 275 280

293 294 367 568 569 583 591 660 662 705 710 815 1746 mz (ES) 336 (MH+) (HRES)

MH+ found 3362383 C16H34NO6

+ requires 3362386

243 General procedure for manual solid-phase oligomerization

PNA oligomers were assembled on a Rink amide PEGA resin using the above obtained Fmoc-

protected PNA modified monomers as well as normal PNA monomers

O

t-BuO

NH2

5

91

O

OH

83

+

O

t-BuO

NR

OHOH

101 R =OH

OH

100 R = H

5

47

Rink amide PEGA resin (50 ndash 100 mg) was first swelled in CH2Cl2 for 30 minutes Normal PNA

monomers (from Link Technologies) was provided by Advance Biosystem Italia srl The Fmoc group

was then deprotected by 20 piperidine in DMF (8 min x 2) The resin was washed with DMF and

CH2Cl2 and was indicated to be positive by the Kaiser test The resin was preloaded with a Fmoc-

Glycine and subsequently the coupling of the monomers (PNA or PNA modified) was conducted with

either one of the following two methods (A and B) Method A monomer (5 eq) HBTU (49 eq) and

DIPEA (10 eq) method B modified monomer (2 eq) HATU (2 eq) and DIPEA (10 eq) When the

monomer was coupled to a primary amine ie to a classic PNA monomer method A was used when

the coupling was to a secondary amine ie to a modified PNA monomer method B was used The

coupling mixture was added directly to the Fmoc-deprotected resin The coupling reaction required 30

minutes at room temperature for the introduction of both normal and modified monomers in case of

method B the coupling time was raised to 6 h and the resin was then washed by DMF CH2Cl2 The

Fmoc deprotection and monomer coupling cycles were continued until the coupling of the last residue

After every coupling the oligomer was capped by adding 5 acetic anhydride and 6 DIPEA in DMF

and the reaction vessel was shaken for 1 min (twice) and subsequently washed with a solution of 5 of

DIPEA in DMF The resin was always washed thoroughly with DMF CH2Cl2 The cleavage from the

resin was achieved by addition of a solution of 90 TFA and 10 m-cresol The PNA was then

precipitated adding 10 volumes of diethyl ether cooled at -18degC for at least 2 h and finally collected

through centrifugation (5 minutes 5000 rpm) The resulting residue was redisolved in H2O and

purified by semipreparative reverse-phase C18 (Waters Bondapak 10 μm 125Aring 78 times 300 mm)

gradient elution from 100 H2O (01 TFA eluent A) to 60 CH3CN (01TFA eluent B) in 30 min

The product was then lyophilized to get a white solid MALDI-TOF MS analysis confirmed the

expected structures for the four oligomers (49-52) with peaks at the following mz values compound 49

mz (MALDI-TOF MS ndash negative mode) 2839 (MndashH)ndash calcd for C112H140N59O33

ndash 283911 60

compound 50 mz (MALDI-TOF MS ndash negative mode) 2955 (MndashH)ndash calcd For C116H144N59O37

ndash

295512 57 compound 51 mz (MALDI-TOF MS ndash negative mode) 2895 (MndashH)ndash calcd for

C116H148N59O33ndash 289517 62 compound 52 mz (MALDI-TOF MS ndash negative mode) 3123 (MndashH)

ndash

calcd for C128H168N59O37ndash 312331 65

244 Thermal denaturation studies

DNA oligonucleotides were purchased from CEINGE Biotecnologie avanzate sc a rl

The PNA oligomers and DNA were hybridized in a buffer 100 mM NaCl 10 mM sodium phosphate

and 01 mM EDTA pH 70 The concentrations of PNAs were quantified by measuring the absorbance

(A260) of the PNA solution at 260 nm The values for the molar extinction coefficients (ε260) of the

individual bases are ε260 (A) = 137 mL(μmole x cm) ε260 (C)= 66 mL(μmole x cm) ε260 (G) = 117

mL(μmole x cm) ε260 (T) = 86 mL(μmole x cm) and molar extinction coefficient of PNA was

calculated as the sum of these values according to sequence

The concentrations of DNA and modified PNA oligomers were 5 μM each for duplex formation The

samples were first heated to 90 degC for 5 min followed by gradually cooling to room temperature

Thermal denaturation profiles (Abs vs T) of the hybrids were measured at 260 nm with an UVVis

Lambda Bio 20 Spectrophotometer equipped with a Peltier Temperature Programmer PTP6 interfaced

to a personal computer UV-absorption was monitored at 260 nm from in a 18-90degC range at the rate of

1 degC per minute A melting curve was recorded for each duplex The melting temperature (Tm) was

determined from the maximum of the first derivative of the melting curves

48

Chapter 3

3 Structural analysis of cyclopeptoids and their complexes

31 Introduction

Many small proteins include intramolecular side-chain constraints typically present as disulfide

bonds within cystine residues

The installation of these disulfide bridges can stabilize three-dimensional structures in otherwise

flexible systems Cyclization of oligopeptides has also been used to enhance protease resistance and cell

permeability Thus a number of chemical strategies have been employed to develop novel covalent

constraints including lactam and lactone bridges ring-closing olefin metathesis76

click chemistry77-78

as

well as many other approaches2

Because peptoids are resistant to proteolytic degradation79

the

objectives for cyclization are aimed primarily at rigidifying peptoid conformations Macrocyclization

requires the incorporation of reactive species at both termini of linear oligomers that can be synthesized

on suitable solid support Despite extensive structural analysis of various peptoid sequences only one

X-ray crystal structure has been reported of a linear peptoid oligomer80

In contrast several crystals of

cyclic peptoid hetero-oligomers have been readily obtained indicating that macrocyclization is an

effective strategy to increase the conformational order of cyclic peptoids relative to linear oligomers

For example the crystals obtained from hexamer 102 and octamer 103 (figure 31) provided the first

high-resolution structures of peptoid hetero-oligomers determined by X-ray diffraction

102 103

Figure 31 Cyclic peptoid hexamer 102 and octamer 103 The sequence of cistrans amide bonds

depicted is consistent with X-ray crystallographic studies

Cyclic hexamer 102 reveals a combination of four cis and two trans amide bonds with the cis bonds

at the corners of a roughly rectangular structure while the backbone of cyclic octamer 103 exhibits four

cis and four trans amide bonds Perhaps the most striking observation is the orientation of the side

chains in cyclic hexamer 102 (figure 32) as the pendant groups of the macrocycle alternate in opposing

directions relative to the plane defined by the backbone

76 H E Blackwell J D Sadowsky R J Howard J N Sampson J A Chao W E Steinmetz D J O_Leary

R H Grubbs J Org Chem 2001 66 5291 ndash5302 77 S Punna J Kuzelka Q Wang M G Finn Angew Chem Int Ed 2005 44 2215 ndash2220

78 Y L Angell K Burgess Chem Soc Rev 2007 36 1674 ndash1689 79 S B Y Shin B Yoo L J Todaro K Kirshenbaum J Am Chem Soc 2007 129 3218 ndash3225

80 B Yoo K Kirshenbaum Curr Opin Chem Biol 2008 12 714 ndash721

49

Figure 32 Crystal structure of cyclic hexamer 102[31]

In the crystalline state the packing of the cyclic hexamer appears to be directed by two dominant

interactions The unit cell (figure 32 bottom) contains a pair of molecules in which the polar groups

establish contacts between the two macrocycles The interface between each unit cell is defined

predominantly by aromatic interactions between the hydrophobic side chains X-ray crystallography of

peptoid octamer 103 reveals structure that retains many of the same general features as observed in the

hexamer (figure 33)

Figure 33 Cyclic octamer 1037 Top Equatorial view relative to the cyclic backbone Bottom Axial

view backbone dimensions 80 x 48 Ǻ

The unit cell within the crystal contains four molecules (figure 34) The macrocycles are assembled

in a hierarchical manner and associate by stacking of the oligomer backbones The stacks interlock to

form sheets and finally the sheets are sandwiched to form the three-dimensional lattice It is notable that

in the stacked assemblies the cyclic backbones overlay in the axial direction even in the absence of

hydrogen bonding

50

Figure 34 Cyclic octamer 103 Top The unit cell contains four macrocycles Middle Individual

oligomers form stacks Bottom The stacks arrange to form sheets and sheets are propagated to form the

crystal lattice

Cyclic α and β-peptides by contrast can form stacks organized through backbone hydrogen-bonding

networks 81-82

Computational and structural analyses for a cyclic peptoid trimer 30 tetramer 31 and

hexamer 32 (figure 35) were also reported by my research group83

Figure 35 Trimer 30 tetramer 31 and hexamer 32 were also reported by my research group

Theoretical and NMR studies for the trimer 30 suggested the backbone amide bonds to be present in

the cis form The lowest energy conformation for the tetramer 31 was calculated to contain two cis and

two trans amide bonds which was confirmed by X-ray crystallography Interestingly the cyclic

81 J D Hartgerink J R Granja R A Milligan M R Ghadiri J Am Chem Soc 1996 118 43ndash50

82 F Fujimura S Kimura Org Lett 2007 9 793 ndash 796 83 N Maulucci I Izzo G Bifulco A Aliberti C De Cola D Comegna C Gaeta A Napolitano C Pizza C

Tedesco D Flot F De Riccardis Chem Commun 2008 3927 ndash3929

51

hexamer 32 was calculated and observed to be in an all-trans form subsequent to the coordination of

sodium ions within the macrocycle Considering the interesting results achieved in these cases we

decided to explore influences of chains (benzyl- and metoxyethyl) in the cyclopeptoidslsquo backbone when

we have just benzyl groups and metoxyethyl groups So we have synthesized three different molecules

a N-benzyl cyclohexapeptoid 56 a N-benzyl cyclotetrapeptoid 57 a N-metoxyethyl cyclohexapeptoid

58 (figure 36)

N

OO

O

N

O

N

O

56

N

NN

OO

O

N

O

57

N

O

N

O ON

N

O

O58

cyclohexapeptoid 58

321 Chemistry

The synthesis of linear hexa- (104) and tetra- (105) N-benzyl glycine oligomers and of linear hexa-

N-metoxyethyl glycine oligomer (106) was accomplished on solid-phase (2-chlorotrityl resin) using the

―sub-monomer approach84

(scheme 31)

52

Cl

HOBr

O

OBr

O

HON

H

O

HON

H

O

n=6 106

n=6 104n=4 105

NH2

ONH2

n

Scheme 31 ―Sub-monomer approach for the synthesis of linear tetra- (104) and hexa- (105) N-

benzyl glycine oligomers and of linear hexa-N-metoxyethyl glycine oligomers (106)

All the reported compounds were successfully synthesized as established by mass spectrometry with

isolated crude yields between 60 and 100 and purities greater than 90 by HPLC analysis85

Head-to-tail macrocyclization of the linear N-substituted glycines were realized in the presence of

PyBop in DMF (figure 37)

HON

NN

O

N

O

NNH

O

N

OO

O

N

O

N

O

PyBOP DIPEA DMF

104

56

80

HON

NN

O

NH

O

N

NN

OO

O

N

O

PyBOP DIPEA DMF

105

57

85 Analytical HPLC analyses were performed on a Jasco PU-2089 quaternary gradient pump equipped with an MD-

2010 plus adsorbance detector using C18 (Waters Bondapak 10 μm 125Aring 39 times 300 mm) reversed phase

columns

53

HON

NN

O

N

O

NNH

O

O O O

O

106

N

O

N

O ON

N

O

O58

PyBOP DIPEA DMF

87

Figure 37 Cyclization of oligomers 104 105 and 106

Several studies in model peptide sequences have shown that incorporation of N-alkylated amino acid

residues can improve intramolecular cyclization86a-b-c

By reducing the energy barrier for interconversion

between amide cisoid and transoid forms such sequences may be prone to adopt turn structures

facilitating the cyclization of linear peptides87

Peptoids are composed of N-substituted glycine units

and linear peptoid oligomers have been shown to readily undergo cistrans isomerization Therefore

peptoids may be capable of efficiently sampling greater conformational space than corresponding

peptide sequences88

allowing peptoids to readily populate states favorable for condensation of the N-

and C-termini In addition macrocyclization may be further enhanced by the presence of a terminal

secondary amine as these groups are known to be more nucleophilic than corresponding primary

amines with similar pKalsquos and thus can exhibit greater reactivity89

322 Structural Analysis

Compounds 56 57 and 58 were crystallized and subjected to an X-ray diffraction analysis For the

X-ray crystallographic studies were used different crystallization techniques like as

1 slow evaporation of solutions

2 diffusion of solvent between two liquids with different densities

3 diffusion of solvents in vapor phase

4 seeding

The results of these tests are reported respectively in the tables 31 32 and 33 above

86 a) Blankenstein J Zhu J P Eur J Org Chem 2005 1949-1964 b) Davies J S J Pept Sci 2003 9 471-

501 c) Dale J Titlesta K J Chem SocChem Commun 1969 656-659 87 Scherer G Kramer M L Schutkowski M Reimer U Fischer G J Am Chem Soc 1998 120 5568-

5574 88 Patch J A Kirshenbaum K Seurynck S L Zuckermann R N In Pseudo-peptides in Drug

DeVelopment Nielson P E Ed Wiley-VCH Weinheim Germany 2004 pp 1-35 89 (a) Bunting J W Mason J M Heo C K M J Chem Soc Perkin Trans 2 1994 2291-2300 (b) Buncel E

Um I H Tetrahedron 2004 60 7801-7825

54

Table 31 Results of crystallization of cyclopeptoid 56

SOLVENT 1 SOLVENT 2 Technique Results

1 CHCl3 Slow evaporation Crystalline

precipitate

2 CHCl3 CH3CN Slow evaporation Precipitate

3 CHCl3 AcOEt Slow evaporation Crystalline

precipitate

4 CHCl3 Toluene Slow evaporation Precipitate

5 CHCl3 Hexane Slow evaporation Little crystals

6 CHCl3 Hexane Diffusion in vapor phase Needlelike

crystals

7 CHCl3 Hexane Diffusion in vapor phase Prismatic

crystals

8 CHCl3

Hexane Diffusion in vapor phase

with seeding

Needlelike

crystals

9 CHCl3 Acetone Slow evaporation Crystalline

precipitate

10 CHCl3 AcOEt Diffusion in

vapor phase

Crystals

11 CHCl3 Water Slow evaporation Precipitate

55

SOLVENT 1 SOLVENT 2 SOLVENT 3 Technique Results

1 CH2Cl2 Slow

evaporation

Prismatic

crystals

2 CHCl3 Slow

evaporation

Precipitate

3 CHCl3 AcOEt CH3CN Slow

evaporation

Crystalline

Aggregates

4 CHCl3 Hexane Slow

evaporation

Little

crystals

1 CHCl3 Slow

evaporation

Crystals

2 CHCl3 CH3CN Slow

evaporation

Precipitate

3 AcOEt CH3CN Slow

evaporation

Precipitate

5 AcOEt CH3CN Slow

evaporation

Prismatic

crystals

6 CH3CN i-PrOH Slow

evaporation

Little

crystals

7 CH3CN MeOH Slow

evaporation

Crystalline

precipitate

8 Esano CH3CN Diffusion

between two

phases

Precipitate

9 CH3CN Crystallin

precipitate

56

Trough these crystallizations we had some crystals suitable for the analysis Conditions 6 and 7

(compound 56 table 31) gave two types of crystals (structure 56A and 56B figure 38)

56A 56B

Figure 38 Structures of N-Benzyl-cyclohexapeptoid 56A and 57B

For compound 57 condition 1 (table 32) gave prismatic crystals (figure 39)

57

Figure 39 Structures of N-Benzyl-cyclotetrapeptoid 57

For compound 58 condition 5 (table 32) gave prismatic colorless crystals (figure 310)

58

Figure 310 Structures of N-metoxyethyl-cyclohexapeptoid 58

57

Table 34 reports the crystallographic data for the resolved structures 56A 56B 57 and 58

Compound 56A 56B 57 58

Formula C54 H54 N6 O6 2H2O C54 H54 N6 O6 C36 H36 N4 O4 C30 H54 N6 O12

PM (g mol-1

) 91903 88303 58869 51336

Dim crist (mm) 07 x 02 x 01 02 x 03 x 008 03 x 03 x 007 03 x 01x 005

Source Rotating

anode

Rotating

anode

Rotating

anode

Rotating

anode

λ (Aring)

154178 154178 154178 154178

Cristalline system monoclinic triclinic orthorhombic triclinic

Space group C2c P Pbca P

a (Aring)

b (Aring)

c (Aring)

α (deg)

β (deg)

γ (deg)

4573(7)

9283(14)

2383(4)

10597(4)

9240(12)

11581(13)

11877(17)

10906(2)

10162(5)

92170(8)

10899(3)

10055(3)

27255(7)

8805(3)

11014(2)

12477(2)

7097(2)

77347(16)

8975(2)

V (Aring3 ) 9725(27) 1169(3) 29869(14) 11131(5)

Z 8 1 4 2

Dcalc (g cm-3

) 1206 1254 1309 1532

58

μ (cm-1

) 0638 0663 0692 2105

Total reflection 7007 2779 2253 2648

Observed

reflecti

on (Igt2I )

4883 1856 1985 1841

R1 (Igt2I) 01345 00958 00586 01165

Rw 04010 03137 02208 03972

323 Structural analysis of N-Benzyl-cyclohexapeptoid 56A

Initially single crystals of N-benzylcyclohexapeptoid 56 were formed by slow evaporation of

solvents (chloroformhexane=105) Optimal conditions of crystallization were in chloroform trough

vapor phase diffusion of hexane (external solvent) and with seeding These techniques gave air stable

needlelike crystals (34A)

The crystals show a monoclinic crystalline cell with the following parameters a = 4573(7) Aring b =

9283(14) Aring c = 2383(4) Aring β = 10597(4)deg V = 9725(27) Aring3 C2c space group Eight molecules of 56

and 4 molecules of water were present in the elementary cell Water molecules are on a binary

symmetry axis and they formed a bridge trough a hydrogen bond with a carbonyl group of

cyclopeptoids between two molecules of equivalent cyclopeptoids These water molecules formed a

water channel (figure 311) The peptoid backbone of 56A showed an overall rectangular form with

four cis amide bonds reside at each corner with two trans amide bonds were present on two opposite

sides

56A

59

View along the axis b

View along the axis c

Figure 311 Hydrogen bond between water and two equivalent cyclopeptoids Blue and pink benzyl group are

pseudo parallel to each other while yellow benzyl group are pseudo perpendicular to each other

324 Structural analysis of N-Benzyl-cyclohexapeptoid 56B

Cyclohexamer 56 was formed two different crystals (6 and 7 table 31) In condition 7 it formed

prismatic crystals and showed a triclinic structure (56B) The cell parameters were a = 9240(12)Aring b =

11581(13)Aring c = 11877(17)Aring α = 10906(2)deg β = 10162(5)deg γ = 92170(8)deg V = 1169(3) Aring3 the

space group is P1

Just cyclopeptoid 56B was into the cell and crystallographic gravity centre was coincident with

inversion centre

60

Monoclinic (56A) and Triclinic (56B) structures of cyclopeptoid 56 showed the same backbone but

benzyl groups had a different orientation In figure 312 is showed the superposition of two structures

Figure 312 superposition of two structures 56A and 56B

Triclinic crystalline cell [a = 9240(12)Aring b = 11581(13)Aring c = 11877(17)Aring α = 10906(2)deg β =

10162(5)deg γ = 92170(8)deg] is been compared with monoclinic cell [a = 4573(7) Aring b = 9283(14) Aring c

= 2383(4) Aring β = 10597(4)deg] and we could see a new triclinic cell similar to the first if we applied the

following operation on triclinic cell

arsquo 0 1 0 a b

brsquo = 0 0 1 b = c

crsquo 1 0 0 c a

a = 11877(17)Aring b = 9240(12)Aring c = 11581(13)Aring α = 92170(8)deg β 10906(2)deg γ= 10162(5)deg so

aM=4 aT bM=bT e cM=2cT

The triclinic cell was considered a distortion of the monoclinic cell In figure 313 were reported the

structure of 56B

View along the axis a

61

View along the axis b View along the axis c

Figure 313 Crystalline structure of 56B

325 Structural analysis of N-Benzyl-cyclotetra peptoid 57

Cyclotetramer 57 was obtained by slowly evaporation of solvent (CH2Cl2) as colorless prismatic and

stable crystals (figure 314) They presented an orthorhombic crystalline cell [a = 10899(3) Aring b =

10055(3) Aring c = 27255(7) Aring V = 29869(14) Aring3 ] and they belonged to space group Pbca

X-Ray structure of 57 showed a cis-trans-cis-trans (ctct) stereochemical arrangement benzyl group

were parallel to each other and two of these were pseudo-equatorial (figure 314)

Figure 314 Crystalline structure of 57

326 Structural analysis of N-metoxyethyl-cyclohexapeptoid 58

Single crystal (58) was obtained in slowly evaporation of solvent (AcOEtACN) as colorless

prismatic and stable crystals They were present triclinic crystalline cell with parameters cell a =

8805(3) Aring b = 11014(2) Aring c = 12477(2) Aring α = 7097(2)deg β = 77347(16)deg γ = 8975(2)deg V =

11131(5) Aring3 and they belonged to space group P

62

1H-NMR studies confirmed the presence of a complex species of 58 and it was analyzed by X-ray

method (figure 315)

Figure 315 X-ray structure of 58

The structure obtained showed a unique all-trans peptoid bond configuration with the carbonyl

groups alternately pointing toward the sodium cation and forcing the N-linked side chains to assume an

alternate pseudo-equatorial arrangement X-ray showed the presence of an anion (hexafluorophosphate)

too Hexafluorophosphate is present as counterion of the coupling agent (PyBop) The structure of 58

was described like a coordination polymer in fact two sodium atoms (figure 316) were coordinated

with a cyclopeptoid and this motif was repeat along the axis a

(a)

63

(b)

Figure 316 (a) View along the axis a (b) perspective view of the rows in the crystalline cell of 58

33 X-ray analysis on powder of 56A and 56B

Polymorphous crystalline structures of 56A and 56B were analyzed to confirm analogy between

polymorphous species Crystals were obtained by tests 6 and 7 (table 31) and were ground in a

mortar until powder The powder was put into capillaries (005 mm) and X-ray spectra were acquired in

a range of 4deg-45deg of 2 X-ray spectra have confirmed crystallinity of the sample as well as his

polymorphism (figure 317)

Figure 317 Diffraction profiles for 56A (a) and 56B (b)

Diffraction profiles acquired were indexed using data on the monoclinic and triclinic unit cell In

particular on the left of spectra peaks were similar for both polymorphs Instead on the right of

spectra were present diffraction peaks typical of one of two species

64

34 Conclusions

In this chapter the synthesis and structural characterization of three cyclopeptoids (56 57 and 58)

were reported

For compound 56 two different polymorph structures were isolated (56A and 56B) crystalline

structure of 56A shows a monoclinic cell and a water channel Instead crystalline structure of 56B

presents a triclinic cell without water channel Backbonelsquos conformation of 56A and 56B is similar

(cctcct) but benzyl groupslsquo conformation is different X-ray analysis on powders of 56A and 56B has

confirmed the non coexistence of the monoclinic and triclinic structures in the same sample N-

benzylcyclotetrapeptoid 57 presents an orthorhombic cell and a conformation ctct

Finally N-metoxyethylcyclohexapeptoid 58 has been isolated as sodium ion complex The

crystalline structure consists of rows of cyclopeptoids and sodium ions hexafluophosphate ions are in

the gaps Backbonelsquos conformation results to be all trans (tttttt) and sodium ions are coordinated with

secondary interactions to the oxygens of carbonyl groups and to the oxygens of methoxy groups

351 General Methods

400 (1H at 40013 MHz

1H at 25013 MHz

13C at 6289 MHz)

CDCl3 = 770 CD2HOD

= 334 13

capillary temperature was 220 degC HPLC analyses were performed on a Jasco BS 997-01 series

65

352 Synthesis

Linear peptoid oligomers 104 105 and 106 were synthesized using a sub-monomer solid-phase

approach11

In a typical synthesis 2-chlorotrityl chloride resin (2α-dichlorobenzhydryl-polystyrene

crosslinked with 1 DVB 100ndash200 mesh 12 mmolg 600 mg 0720 mmol) was swelled in dry DMF

(6 mL) for 45 min and washed twice with dry DCM (6 mL)

The first sub-monomer was attached onto the resin by adding 120 mg (12 eq 086 mmol) of

bromoacetic acid in dry DCM (6 mL) and 502 μL of DIPEA (40 mmol) on a shaker platform for 40 min

at room temperature followed by washing with dry DCM (6 mL) and then with DMF (6 x 6 mL) To the

bromoacetylated resin was added a DMF solution (1 M 7 mL) of the desired amine (benzylamine -10

eq 772 mg 720 mmol- or methoxyethylamine -10 eq 541 mg 620 μl 720 mmol- the commercially

available [Aldrich]) the mixture was left on a shaker platform for 30 min at room temperature then the

resin was washed with DMF (6 x 6 mL) Subsequent bromoacetylation reactions were accomplished by

reacting the aminated oligomer with a solution of bromoacetic acid in DMF (12 M 6 mL) and 123 mL

of DIC for 40 min at room temperature The filtered resin was washed with DMF (6 x 6 mL) and treated

again with the amine in the same conditions reported above This cycle of reactions was iterated until

the target oligomer was obtained (hexa- 104 and tetralinears 105 and 106 peptoids)

The cleavage was performed by treating twice the resin previously washed with DCM (6 x 6 mL)

with 6 mL of 20 HFIP in DCM (vv) on a shaker platform at room temperature for 30 min and 5 min

respectively The resin was then filtered away and the combined filtrates were concentrated in vacuo

The final products were dissolved in 50 acetonitrile in HPLC grade water and analysed by RP-HPLC

(purity gt95 for all the oligomers conditions 5 to 100 B in 30 min [A 01 TFA in water B

01 TFA in acetonitrile] flow 10 mLmin 220 nm C18 reversed-phase analytical column [Waters

μBondapak 10 lm 125 Aring 39 mm x 300 mm]) and ESI mass spectrometry The linear oligomers 75 76

and 77 were subjected to the cyclization reaction without further purification

Compound 104 mz (ES) 901 (MH+) (HRES) MH

+ found 9014290 C54H57N6O7

+ requires

9014289 100

+ found 6072925 C36H39N4O5

+ requires

6062920 100

+ found 7093986 C30H57N6O13

+ requires

7093984 100

353 General cyclization reaction (synthesis of 56 57 and 58)

A solution of the linear peptoid (104 105 and 106) previously co-evaporated three times with

toluene was prepared under nitrogen in dry DMF (20 mL)

66

Linear 104 (37 mg 00611 mmol) was dissolved in DMF dry (5 mL) and the mixture was

added drop-wise by syringe pump in 2 h to a stirred solution of PyBop (127 mg 4 eq 0244 mmol) and

DIPEA (49 mg 62 eq 66 μl 0319 mmol) in dry DMF (15 mL) at room temperature in anhydrous

atmosphere

added drop-wise by syringe pump in 2 h to a stirred solution of PyBop (40 eq 692 mg 137 mmol) and

DIPEA (60 eq 360 microl 206 mmol) in dry DMF (20 mL) at room temperature in anhydrous atmosphere

atmosphere

After 12 h the resulting mixtures were concentrated in vacuo diluted with CH2Cl2 (20 mL) and a

solution of HCl (1 N 20 mL) The single mixture was extracted with CH2Cl2 (2 x 20 mL) and the

combined organic phases were washed with water (12 mL) dried over anhydrous Na2SO4 filtered and

concentrated in vacuo

All cyclic products were dissolved in 50 acetonitrile in HPLC grade water and analysed by RP-

HPLC (purity gt85 for all the cyclic oligomers)

Eluition conditions 5-100 B in 30 min [A 01 TFA in water B 01 TFA in acetonitrile]

flow 10 mLmin 220 nm C18 reversed-phase analytical column [Waters lBondapak 10 lm 125 Aring

39 mm x 300 mm] and ESI mass spectrometry (zoom scan technique)

The crude residues (56 57 and 58) were purified by HPLC on a C18 reversed-phase preparative

column conditions 20-100 B in 40 min [A 01 TFA in water B 01 TFA in acetonitrile] flow

20 mLmin 220 nm The samples were dried in a falcon tube under low pressure

Compound 56 δH (30010 MHz CDC13) 715 (30 H m H Ar) 440 (12H br -NCHHCO -

CH2Ph) 380 (4H br -CH2Ph) 338 (8H m -CH2Ph) 80 mz (ES) 883 (MH+) (HRES) MH

+ found

8834110 C54H55N6O6+

requires 8824105 HPLC tR 199 min

Compound 57 H ( 250 MHz CDCl3) 723 (20 H br H Ar ) 555 (2 H br d J 14 Hz -

NCHHCO) 540 (2 H br d J 145 Hz -NCHHCO) 440 (6 H m J 17 Hz -CH2Ph) 372 (2 H br d

J 142 Hz -NCHHCO) 350 (4 H br d J 145 Hz -NCHHCO -CH2Ph) C (75 MHz CDCl3) 16894

(CO x 2) 16778 (CO x 2) 13572 (Cq-Ar x 2) 13515 (Cq-Ar x 2) 12891 (C-Ar x 8) 12856 ( C-Ar x

4) 12782 (C-Ar x 4) 12752 ( C-Ar x 4) 5029 ( C x 2) 4930 (C x 2) 4882 (C x 2) 4714 (C x 2)

57 mz (ES) 589 (MH+) (HRES) MH

+ found 5892740 C36H37N4O4

+ requires 5892737 HPLC tR

180 min

Compound 58 H (250 MHz CDCl3) 463 (3 H br d J 17 Hz -NCHHCO) 388 (3 H br

d J 17 Hz -NCHHCO) 348 (48 H m -NCHHCO -CH2CH2OCH3) C (400 MHz CDCl3 mixture of

rotamers) 171 5 1710 1796 1701 1700 1698 1697 1695 1693 1691 1689 1687 1682

1681 717 715 714 713 710 708 706 (bs) 702 (bs) 700 (bs) 698 (bs)695 (bs) 693 (bs)

691 (bs) 688 (bs) 687 (bs) 591 (bs) 589 (bs) 586 (bs) 582 (bs) 534 (bs) 524 (bs) 522 (bs)

67

480 (bs) 476 (bs) 474 (bs)470 (bs) 468 (bs) 466 (bs) 459 (bs) 455 (bs) 433 (bs) 87 mz

+ found 6913810 C30H55N6O12

+ requires 6913800 HPLC tR 118 min

354 General method of X-ray analysis

X-ray analysis were made with Bruker D8 ADVANCE utilized glass capillaries Lindemann and

diameters of 05 mm CuKα was used as radiations with wave length collimated (15418 Aring) and

parallelized using Goumlbel Mirror Ray dispersion was minimized with collimators (06 - 02 - 06) mm

Below I report diffractometric on powders analysis of 56A and 56B

X-ray analysis on powders obtained by crystallization tests

Crystals were obtained by crystallization 6 of 56 they were ground into a mortar and introduced

into a capillary of 05 mm Spectra was registered on rotating capillary between 2 = 4deg and 2 = 45deg

the measure was performed in a range of 005deg with a counting time of 3s In a similar way was

analyzed crystal 7 of 56

X-ray analysis on single crystal of 56A

56A was obtained by 6 table 3 in chloroform with diffusion in vapor phase of hexane (extern

solvent) and subsequently with seeding These crystals were needlelike and air stable A crystal of

dimension of 07 x 02 x 01 mm was pasted on a glass fiber and examined to room temperature with a

diffractometer for single crystal Rigaku AFC11 and with a detector CCD Saturn 944 using a rotating

anode of Cu and selecting a wave length CuKα (154178 Aring) Elementary cell was monoclinic with

parameters a = 4573(7) Aring b = 9283(14) Aring c = 2383(4) Aring β = 10597(4)deg V = 9725(27) Aring3 Z=8 and

belonged to space group C2c

Data reduction

7007 reflections were measured 4883 were strong (F2 gt2ζ (F2 )) Data were corrected for Lorentz

and polarization effects The linear absorption coefficient for CuKα was 0638 cm-1 without correction

Resolution and refinement of the structure

Resolution program was called SIR200290 and it was based on representations theory for evaluation

of the structure semivariant in 1 2 3 and 4 phases It is more based on multiple solution technique and

on selection of most probable solutions technique too The structure was refined with least-squares

techniques using the program SHELXL9791

Function minimized with refinement is 222

0)(

cFFw

considering all reflections even the weak

The disagreement index that was optimized is

2

0

22

0

2

iii

iciii

Fw

FFwwR

90 SIR92 A Altomare et al J Appl Cryst 1994 27435 91 G M Sheldrick ―A program for the Refinement of Crystal Structure from Diffraction Data Universitaumlt

Goumlttingen 1997

68

It was based on squares of structure factors typically reported together the index R1

Considering only strong reflections (Igt2ζ(I))

The corresponding disagreement index RW2 calculating all the reflections is 04 while R1 is 013

For thermal vibration was used an anisotropic model Hydrogen atoms were collocated in canonic

positions and were included into calculations

Rietveld analysis

Rietveld method represents a structural refinement technique and it use the continue diffraction

profile of a spectrum on powders92

Refinement procedure consists in least-squares techniques using GSAS93 like program

This analysis was conducted on diffraction profile of monoclinic structure 34A Atomic parameters

of structural model of single crystal were used without refinement Peaks profile was defined by a

pseudo Voigt function combining it with a special function which consider asymmetry This asymmetry

derives by axial divergence94 The background was modeled manually using GUFI95 like program Data

were refined for parameters cell profile and zero shift Similar procedure was used for triclinic structure

56B

X-ray analysis on single crystal of 56B

56B was obtained by 7 table 31 by chloroform with diffusion in vapor phase of hexane (extern

solvent) These crystals were colorless prismatic and air stable A crystal (dimension 02 x 03 x 008

mm) was pasted on a glass fiber and was examined to room temperature with a diffractometer for single

crystal Rigaku AFC11 and with a detector CCD Saturn 944 using a rotating anode of Cu and selecting a

wave length CuKα (154178 Aring) Elementary cell was triclinic with parameters a = 9240(12) Aring b =

11581(13) Aring c = 11877(17) Aring α = 10906(2)deg β = 10162(5)deg γ = 92170(8)deg V = 1169(3) Aring3 Z = 1

and belonged to space group P1

Data reduction

92

A Immirzi La diffrazione dei cristalli Liguori Editore prima edizione italiana Napoli 2002 93

A C Larson and R B Von Dreele GSAS General Structure Analysis System LANL Report

LAUR 86 ndash 748 Los Alamos National Laboratory Los Alamos USA 1994 94

P Thompson D E Cox and J B Hasting J Appl Crystallogr1987 20 79 L W Finger D E

Cox and A P Jephcoat J Appl Crystallogr 1994 27 892 95

R E Dinnebier and L W Finger Z Crystallogr Suppl 1998 15 148 present on

wwwfkfmpgdelXrayhtmlbody_gufi_softwarehtml

0

1

ii

icii

F

FFR

69

positions and included into calculations

X-ray analysis on single crystal of 57

57 was obtained by 1 table 32 by slowly evaporation of dichloromethane These crystals were

colorless prismatic and air stable A crystal of dimension of 03 x 03 x 007 mm was pasted on a glass

fiber and was examined to room temperature with a diffractometer for single crystal Rigaku AFC11 and

with a detector CCD Saturn 944 using a rotating anode of Cu and selecting a wave length CuKα

(154178 Aring) Elementary cell is triclinic with parameters a = 10899(3) Aring b = 10055(3) Aring c =

27255(7) Aring V = 29869(14) Aring3 Z=4 and belongs to space group Pbca

Data reduction

For thermal vibration is used an anisotropic model Hydrogen atoms were collocated in canonic

58 was obtained by 5 table 5 by slowly evaporation of ethyl acetateacetonitrile These crystals

were colorless prismatic and air stable A crystal (dimension 03 x 01 x 005mm) was pasted on a glass

(154178 Aring)

Elementary cell was triclinic with parameters a = 8805(3) Aring b = 11014(2) Aring c = 12477(2) Aring α =

7097(2)deg β = 77347(16)deg γ = 8975(2)deg V = 11131(5) Aring3 Z=2 and belonged to space group P1

Data reduction

70

Chapter 4

41 Introduction

Viral and nonviral gene transfer systems have been under intense investigation in gene therapy for

the treatment and prevention of multiple diseases96

Nonviral systems potentially offer many advantages

over viral systems such as ease of manufacture safety stability lack of vector size limitations low

immunogenicity and the modular attachment of targeting ligands97

Most nonviral gene delivery

systems are based on cationic compoundsmdash either cationic lipids2 or cationic polymers

98mdash that

spontaneously complex with a plasmid DNA vector by means of electrostatic interactions yielding a

condensed form of DNA that shows increased stability toward nucleases

Although cationic lipids have been quite successful at delivering genes in vitro the success of these

compounds in vivo has been modest often because of their high toxicity and low transduction

efficiency

A wide variety of cationic polymers have been shown to mediate in vitro transfection ranging from

proteins [such as histones99

and high mobility group (HMG) proteins100

] and polypeptides (such as

polylysine3101

short synthetic peptides102103

and helical amphiphilic peptides104105

) to synthetic

polymers (such as polyethyleneimine106

cationic dendrimers107108

and glucaramide polymers109

)

Although the efficiencies of gene transfer vary with these systems a large variety of cationic structures

are effective Unfortunately it has been difficult to study systematically the effect of polycation

structure on transfection activity

96 Mulligan R C (1993) Science 260 926ndash932 97 Ledley F D (1995) Hum Gene Ther 6 1129ndash1144 98 Wu G Y amp Wu C H (1987) J Biol Chem 262 4429ndash4432 99 Fritz J D Herweijer H Zhang G amp Wolff J A Hum Gene Ther 1996 7 1395ndash1404 100 Mistry A R Falciola L L Monaco Tagliabue R Acerbis G Knight A Harbottle R P Soria M

Bianchi M E Coutelle C amp Hart S L BioTechniques 1997 22 718ndash729 101 Wagner E Cotten M Mechtler K Kirlappos H amp Birnstiel M L Bioconjugate Chem 1991 2 226ndash231 102Gottschalk S Sparrow J T Hauer J Mims M P Leland F E Woo S L C amp Smith L C Gene Ther

1996 3 448ndash457 103 Wadhwa M S Collard W T Adami R C McKenzie D L amp Rice K G Bioconjugate Chem 1997 8 81ndash

88 104 Legendre J Y Trzeciak A Bohrmann B Deuschle U Kitas E amp Supersaxo A Bioconjugate Chem

1997 8 57ndash63 105 Wyman T B Nicol F Zelphati O Scaria P V Plank C amp Szoka F C Jr Biochemistry 1997 36 3008ndash

3017 106 Boussif O Zanta M A amp Behr J-P Gene Ther 1996 3 1074ndash1080 107 Tang M X Redemann C T amp Szoka F C Jr Bioconjugate Chem 1996 7 703ndash714 108 Haensler J amp Szoka F C Jr Bioconjugate Chem 1993 4 372ndash379 109 Goldman C K Soroceanu L Smith N Gillespie G Y Shaw W Burgess S Bilbao G amp Curiel D T

Nat Biotech 1997 15 462ndash466

71

Since the first report in 1987110

cell transfection mediated by cationic lipids (Lipofection figure 41)

has become a very useful methodology for inserting therapeutic DNA into cells which is an essential

step in gene therapy111

Several scaffolds have been used for the synthesis of cationic lipids and they include polymers112

dendrimers113

nanoparticles114

―gemini surfactants115

and more recently macrocycles116

Figure 41 Cell transfection mediated by cationic lipids

It is well-known that oligoguanidinium compounds (polyarginines and their mimics guanidinium

modified aminoglycosides etc) efficiently penetrate cells delivering a large variety of cargos16b117

Ungaro et al reported21c

that calix[n]arenes bearing guanidinium groups directly attached to the

aromatic nuclei (upper rim) are able to condense plasmid and linear DNA and perform cell transfection

in a way which is strongly dependent on the macrocycle size lipophilicity and conformation

Unfortunately these compounds are characterized by low transfection efficiency and high cytotoxicity

110 Felgner P L Gadek T R Holm M Roman R Chan H W Wenz M Northrop J P Ringold G M

Danielsen M Proc Natl Acad Sci USA 1987 84 7413ndash7417 111 (a) Zabner J AdV Drug DeliVery ReV 1997 27 17ndash28 (b) Goun E A Pillow T H Jones L R

Rothbard J B Wender P A ChemBioChem 2006 7 1497ndash1515 (c) Wasungu L Hoekstra D J Controlled

Release 2006 116 255ndash264 (d) Pietersz G A Tang C-K Apostolopoulos V Mini-ReV Med Chem 2006 6

1285ndash1298 112 (a) Haag R Angew Chem Int Ed 2004 43 278ndash282 (b) Kichler A J Gene Med 2004 6 S3ndashS10 (c) Li

H-Y Birchall J Pharm Res 2006 23 941ndash950 113 (a) Tziveleka L-A Psarra A-M G Tsiourvas D Paleos C M J Controlled Release 2007 117 137ndash

146 (b) Guillot-Nieckowski M Eisler S Diederich F New J Chem 2007 31 1111ndash1127 114 (a) Thomas M Klibanov A M Proc Natl Acad Sci USA 2003 100 9138ndash9143 (b) Dobson J Gene

Ther 2006 13 283ndash287 (c) Eaton P Ragusa A Clavel C Rojas C T Graham P Duraacuten R V Penadeacutes S

IEEE T Nanobiosci 2007 6 309ndash317 115 (a) Kirby A J Camilleri P Engberts J B F N Feiters M C Nolte R J M Soumlderman O Bergsma

M Bell P C Fielden M L Garcıacutea Rodrıacuteguez C L Gueacutedat P Kremer A McGregor C Perrin C

Ronsin G van Eijk M C P Angew Chem Int Ed 2003 42 1448ndash1457 (b) Fisicaro E Compari C Duce E

DlsquoOnofrio G Roacutez˙ycka- Roszak B Wozacuteniak E Biochim Biophys Acta 2005 1722 224ndash233 116 (a) Srinivasachari S Fichter K M Reineke T M J Am Chem Soc 2008 130 4618ndash4627 (b) Horiuchi

S Aoyama Y J Controlled Release 2006 116 107ndash114 (c) Sansone F Dudic` M Donofrio G Rivetti C

Baldini L Casnati A Cellai S Ungaro R J Am Chem Soc 2006 128 14528ndash14536 (d) Lalor R DiGesso

J L Mueller A Matthews S E Chem Commun 2007 4907ndash4909 117 (a) Nishihara M Perret F Takeuchi T Futaki S Lazar A N Coleman A W Sakai N Matile S

Org Biomol Chem 2005 3 1659ndash1669 (b) Takeuchi T Kosuge M Tadokoro A Sugiura Y Nishi M

Kawata M Sakai N Matile S Futaki S ACS Chem Biol 2006 1 299ndash303 (c) Sainlos M Hauchecorne M

Oudrhiri N Zertal-Zidani S Aissaoui A Vigneron J-P Lehn J-M Lehn P ChemBioChem 2005 6 1023ndash

1033 (d) Elson-Schwab L Garner O B Schuksz M Crawford B E Esko J D Tor Y J Biol Chem 2007

282 13585ndash13591 (e) Wender P A Galliher W C Goun E A Jones L R Pillow T AdV Drug ReV 2008

60 452ndash472

72

especially at the vector concentration required for observing cell transfection (10-20 μM) even in the

presence of the helper lipid DOPE (dioleoyl phosphatidylethanolamine)16c118

Interestingly Ungaro et al found that attaching guanidinium (figure 42 107) moieties at the

phenolic OH groups (lower rim) of the calix[4]arene through a three carbon atom spacer results in a new

class of cytofectins16

Figure 42 Calix[4]arene like a new class of cytofectines

One member of this family (figure 42) when formulated with DOPE performed cell transfection

quite efficiently and with very low toxicity surpassing a commercial lipofectin widely used for gene

delivery Ungaro et al reported in a communication119

the basic features of this new class of cationic

lipids in comparison with a nonmacrocyclic (gemini-type 108) model compound (figure 43)

108

Figure 43 Nonmacrocyclic cationic lipids gemini-type

The ability of compounds 107 a-c and 108 to bind plasmid DNA pEGFP-C1 (4731 bp) was assessed

through gel electrophoresis and ethidium bromide displacement assays11

Both experiments evidenced

that the macrocyclic derivatives 107 a-c bind to plasmid more efficiently than 108 To fully understand

the structurendashactivity relationship of cationic polymer delivery systems Zuckermann120

examined a set

of cationic N-substituted glycine oligomers (NSG peptoids) of defined length and sequence A diverse

set of peptoid oligomers composed of systematic variations in main-chain length frequency of cationic

118 (a) Farhood H Serbina N Huang L Biochim Biophys Acta 1995 1235 289ndash295 119 Bagnacani V Sansone F Donofrio G Baldini L Casnati A Ungaro R Org Lett 2008 Vol 10 No 18

3953-3959 120 J E Murphy T Uno J D Hamer F E Cohen V Dwarki R N Zuckermann Proc Natl Acad Sci Usa

1998 Vol 95 Pp 1517ndash1522 Biochemistry

73

side chains overall hydrophobicity and shape of side chain were synthesized Interestingly only a

small subset of peptoids were found to yield active oligomers Many of the peptoids were capable of

condensing DNA and protecting it from nuclease degradation but only a repeating triplet motif

(cationic-hydrophobic-hydrophobic) was found to have transfection activity Furthermore the peptoid

chemistry lends itself to a modular approach to the design of gene delivery vehicles side chains with

different functional groups can be readily incorporated into the peptoid and ligands for targeting

specific cell types or tissues can be appended to specific sites on the peptoid backbone These data

highlight the value of being able to synthesize and test a large number of polymers for gene delivery

Simple analogies to known active peptides (eg polylysine) did not directly lead to active peptoids The

diverse screening set used in this article revealed that an unexpected specific triplet motif was the most

active transfection reagent Whereas some minor changes lead to improvement in transfection other

minor changes abolished the capability of the peptoid to mediate transfection In this context they

speculate that whereas the positively charged side chains interact with the phosphate backbone of the

DNA the aromatic residues facilitate the packing interactions between peptoid monomers In addition

the aromatic monomers are likely to be involved in critical interactions with the cell membrane during

transfection Considering the interesting results reported we decided to investigate on the potentials of

cyclopeptoids in the binding with DNA and the possible impact of the side chains (cationic and

hydrophobic) towards this goal Therefore we have synthesized the three cyclopeptoids reported in

figure 44 (62 63 and 64) which show a different ratio between the charged and the nonpolar side

chains

N

NN

N

NNO

O

OO

O

H3N

2X CF3COO-

N

NN

N

O

H3N

NH3

H3N

4X CF3COO-

62 63

74

N

NN

N

NNO

O

OO

O

H3N

NH3

H3N

NH3

6X CF3COO-

64

Figure 44 Dicationic cyclohexapeptoid 62 tetracationic cyclohexapeptoid 63 hexacationic

cyclohexapeptoid 64

421 Synthesis

In order to obtain our targets first step was the synthesis of the amine submonomer N-t-Boc-16-

diaminohexane 110 as reported in scheme 41121

NH2

CH3OH Et3N

NH2

NH

O

110

111

O O O

O O

(Boc)2O

Scheme 41 N-Boc protection

The synthesis of the three N-protected linear precursors (112 113 and 114 scheme 42) was

accomplished on solid-phase (2-chlorotrityl resin) using the ―sub-monomer approach

Cl

HOBr

O

OBr

O

NH2

NH2BocHN

111

121 Krapcho A P amp Kuell C S Synth Commun 1990 20 2559ndash2564

75

HON

O

N

ONHBoc6

N

H

ONHBoc

6

2

N

H

ONHBoc

6

6HO

113

114

HON

O

N

O

N

H

ONHBoc

6

2112

Scheme 42 ―Sub-monomer approach for the synthesis of hexa-linears (112 113 and 114)

Head-to-tail macrocyclizations of the linear N-substituted glycines were realized in the presence of

HATU in DMF according to our previous results122

Cyclization of oligomers 112 113 and 114 proved

to be quite efficient (115 116 and 117 were characterized with HPLC and EI-MS scheme 43)

HON

O

N

O

NH

ONHBoc

6

2

112

HATU DIPEA

DMF 33N

NN

N

NN

O O

O

OO

O

NHO

O

HN

O

115

122 N Maulucci I Izzo G Bifulco A Aliberti C De Cola D Comegna C Gaeta A Napolitano C Pizza C

76

HON

O

N

ONHBoc6

NH

ONHBoc6

2

113

N

NN

N

O

HN

NH

O

OO

O

HN

O O

116

HATU DIPEA

DMF 33

NH

ONHBoc6

6HO114

N

NN

N

NNO

O

OO

O

HNNH

HN

OO

NHO

O

NH

O

NH

O

O O

O

117

HATU DIPEA

DMF 24

Scheme 43 Protected cyclopeptoids 115 116 and 117

All cyclic products were deprotected with 33 TFA in CH2Cl2 to give quantitative yields of

cyclopeptoids 62 63 and 64

422 Biological tests

In collaboration with Donofriolsquos group biological activity evaluation was performed All

cyclopeptoids synthesized for this study should complex spontaneously plasmid DNA owing to an

extensive network of electrostatic interactions The binding of the cationic peptoid to plasmid DNA

should result in neutralization of negative charges in the phosphate backbone of DNA This interaction

can be measured by the inability of the large electroneutral complexes obtained to migrate toward the

cathode during electrophoresis on agarose gel The ability of peptoids to complex with DNA was

evaluated by incubating peptoids with plasmid DNA at a 31 (+-) charge ratio and analyzing the

complexes by agarose gel electrophoresis Peptoids tested in this experiment were not capable of

completely retarding the migration of DNA into the gel In this experiment cyclopeptoids 62 63 and 64

failed to retard the migration of DNA into the gel Therefore the spacing of charged residues on the

77

peptoid backbone as well as the degree of hydrophobicity of the side chains have a dramatic effect on

the ability to form homogenous complexes with DNA in high yield

43 Conclusions

In this project three different cyclopeptoids 62 63 and 64 decorated with cationic side chains were

synthetized Biological evaluation demonstrated that they were not able to act as DNA vectors A

possible reason of these results could be that spatial orientation (see in chapter 3) of the side chains in

cyclopeptoids did not assure the correct coordination and the binding with DNA

441 Synthesis

Compound 111

Di-tert-butyl carbonate (04 eq 751g 0034 mmol) was added to 16-diaminohexane (10 g 0086

mmol) in CH3OHEt3N (91) and the reaction was stirred overnight The solvent was evaporated and the

residue was dissolved in DCM (20 mL) and extracted using a saturated solution of NaHCO3 (20 mL)

Then water phase was washed with DCM (3 middot 20 ml) The combined organic phase was dried over

Mg2SO4 and concentrated in vacuo to give a pale yellow oil The compound was purified by flash

chromatography (CH2Cl2CH3OHNH3 20M solution in ethyl alcohol from 100001 to 703001) to

give 81 (051 g 30) as a yellow light oil [Found C 611 H 112 N 129 O148 C11H24N2O2

requires C 6107 H 1118 N 1295 O 1479] Rf (98201 CH2Cl2CH3OHNH3 20M solution in

ethyl alcohol) 063 δH (250 MHz CDCl3) 484 (brs 1H NH-Boc) 297-294 (bq 2H CH2-NH-Boc

J = 75 MHz) 256-251 (t 2H CH2NH2 J 70 MHz) 18 (brs 2H NH2) 130 (s 9H (CH3)3)

130-118 (m 8H CH2) mz (ES) 217 (MH+) (HRES) MH

+ found 2171920 C11H25N2O2

+ requires

2171916

442 General procedures for linear oligomers 112 113 and 114

approach11

crosslinked with 1 DVB 100ndash200 mesh 133 mmolg 400 mg 0532 mmol) was swelled in dry

DMF (4 mL) for 45 min and washed twice with dry DCM (4 mL)

bromoacetic acid in dry DCM (4 mL) and 370 μL of DIPEA (210 mmol) on a shaker platform for 40

min at room temperature followed by washing with dry DCM (4 mL) and then with DMF (4 x 4 mL)

To the bromoacetylated resin was added a DMF solution (1 M 53 mL) of the desired amine

(benzylamine -10 eq- or 111 -10 eq-) the mixture was left on a shaker platform for 30 min at room

temperature then the resin was washed with DMF (4 x 4 mL) Subsequent bromoacetylation reactions

were accomplished by reacting the aminated oligomer with a solution of bromoacetic acid in DMF (12

M 53 mL) and 823 μL of DIC for 40 min at room temperature The filtered resin was washed with

DMF (4 x 4 mL) and treated again with the amine in the same conditions reported above This cycle of

reactions was iterated until the target oligomer was obtained (112 113 and 114 peptoids) The cleavage

was performed by treating twice the resin previously washed with DCM (6 x 6 mL) with 4 mL of 20

HFIP in DCM (vv) on a shaker platform at room temperature for 30 min and 5 min respectively The

78

resin was then filtered away and the combined filtrates were concentrated in vacuo The final products

were dissolved in 50 acetonitrile in HPLC grade water and analysed by RP-HPLC (purity gt95 for

all the oligomers conditions 5 to 100 B in 30 min [A 01 TFA in water B 01 TFA in

acetonitrile] flow 10 mLmin 220 nm C18 reversed-phase analytical column [Waters μBondapak 10

μm 125 Aring 39 mm x 300 mm]) and ESI mass spectrometry The linear oligomers 112 113 and 114

were subjected to the cyclization reaction without further purification

Compound 112 tR196 min mz (ES) 1119 (MH+) (HRES) MH

+ found 11196485

C62H87N8O11+ requires 11196489 100

+ found 13378690

C70H117N10O15+ requires 13378694 100

+ found 15560910

C78H147N12O19+ requires 15560900 100

Linear 112 (10 eq 104 mg 0093 mmol) was dissolved in DMF dry (10 mL) and the

mixture was added drop-wise by syringe pump in 2 h to a stirred solution of HATU (31 eq 1120 mg

029 mmol) and DIPEA (60 eq 97 microl 055 mmol) in dry DMF (20 mL) at room temperature in

anhydrous atmosphere

050 mmol) and DIPEA (60 eq 1746 microl 100 mmol ) in dry DMF (20 mL) at room temperature in

All protected cyclic 115 116 and 117 were dissolved in 50 acetonitrile in HPLC grade water and

analysed by RP-HPLC (purity gt85 for all the cyclic oligomers conditions 5-100 B in 30 min [A

01 TFA in water B 01 TFA in acetonitrile] flow 10 mLmin 220 nm C18 reversed-phase

analytical column [Waters μBondapak 10 μm 125 Aring 39 mm x 300 mm]) and ESI mass spectrometry

(zoom scan technique)

79

The crude residues were purified by HPLC on a C18 reversed-phase preparative column conditions

20-100 B in 40 min [A 01 TFA in water B 01 TFA in acetonitrile] flow 20 mLmin 220

nm The samples were dried in a falcon tube under low pressure

Compound 115 δH (400 MHz CD3CNCDCl3 (91) mixture of conformers) 734-706 (m

20H H-Ar) 519 (br s 2H NH) 480-360 (m 20H NCH2Ph -COCH2N- overlapped) 333-309 (m

4H -CH2N) 302-296 (m 4H -CH2NHBoc) 138 (br s 18 H C(CH3)3) 138-117 (m 16 H (CH2)4)

33 mz (ES) 1101 (MH+) (HRES) MH

+ found 11013785 C62H85N8O10

+ requires 11013780 HPLC

tR 206 min

Compound 116 δH (400 MHz CD3OD mixture of conformers) 746-732 (m 10H H-Ar)

490-320 (m 24H NCH2Ph -COCH2N- -CH2N overlapped with HDO e MeOD) 307-284 (m 8H -

CH2NHBoc) 148 (br s 36 H C(CH3)3) 172-117 (m 32 H (CH2)4) δC (100 MHz CDCl3 mixture of

conformers) 1706 1690 1689 15863 1561 1558 1557 1444 1434 1433 1408 1407 1362

1360 1279 1275 1274 1269 1264 1245 1194 817 816 675 670 660 659 598 504

500 499 487 483 467 466 390 274 205 33 mz (ES) 1319 (MH+) (HRES) MH

+ found

13197130 C70H115N10O14+ requires 13197128 HPLC tR 212 min

Compound 117 δH (250 MHz CD3OD mixture of conformers) 490-320 (m 24H -

COCH2N--CH2N overlapped with HDO e MeOD) 310-285 (m 12H -CH2NHBoc) 144 (br s 54 H

C(CH3)3) 172-120 (m 48 H (CH2)4) δC (625 MHz CD3OD mixture of conformers) 1735 (bs)

1723 (bs) 1719 (bs) 1717 (bs) 1712 (bs) 1709 (bs) 1706 (bs) 1702 (bs) 1585 797 506 (bs)

500 - 480 (overlapped with MeOD) 412 406 (bs) 309 297 (bs) 294 (bs) 288 276 (bs) 24

+ found 15380480 C78H145N12O18

225 min

444 General deprotection reaction (synthesis of 62 63 and 64)

Protected cyclopeptoids 115 (34 mg 0030 mmol) 116 (389 mg 0029 mmol) and 117 (26 mg

0017mmol) were dissolved in a mixture of DCM and TFA (91 4 mL) and the reaction was stirred for

two hours After products were precipitated in cold Ethyl Ether (20 mL) and centrifuged Solids were

recuperated with a quantitative yield

Compound 62 δH (250 MHz MeOD mixture of conformers) 738-701 (m 20H H-Ar) 480

- 330 (m 24H NCH2Ph -COCH2N -CH2N overlapped with HDO and MeOD) 300-283 (m 4H -

CH2NH3+) 175 -130 (m 16 H (CH2)4) δC (75 MHz MeOD mixture of conformers) 1742 (bs)

1721 (bs) 1715 (bs) 1711 (bs) 1710 (bs) 1622 (bs CF3COO-) 1382 (bs) 1380 (bs) 1375 (bs)

1371 (bs) 1311 (bs) 1302 1297 1293 1290 1286 1283 1270 548 538 528 521 (bs) 508

(bs) 506 498-481 (overlapped with MeOD) 406 307 285 281 271 242 mz (ES) 916 (MH+)

3+ requires 9161797

490 - 330 (m 24H NCH2Ph -COCH2N -CH2N overlapped with HDO and MeOD) 300-283 (m

80

8H -CH2NH3+) 175 -130 (m 32 H (CH2)4) mz (ES) 923 (MH

+) (HRES) MH

+ found 9232792

C50H87N10O65+

requires 9232792

Compound 64 δH(300 MHz CD3OD mixture of conformers) 490-316 (m 24H -

COCH2N- -CH2N overlapped with HDO and MeOD) 310-289 (m 12H -CH2NH3+) 172-125 (m

48 H (CH2)4 mz (ES) 943 (MH+) (HRES) MH

+ found 9433978 C48H103N12O6

7+ requires 9433970

445 DNA preparation and storage

Plasmid DNA was purified through cesium chloride gradient centrifugation (T Maniatis EF

Fritsch J Sambrook in Molecular Cloning A Laboratory Manual 2nd edition Cold Spring Harbor

Laboratory New York 1989) A stock solution of the plasmid 0350 M in milliQ water (Millipore

Corp Burlington MA) was stored at -20 degC

446 Electrophoresis mobility shift assay (EMSA)

Binding reactions were performed in a final volume of 14 microL with 10 microl of 20 mM TrisHCl pH 8 1

microL of plasmid (1 microg of pEGFP-C1) and 3 microL of compound 62 63 and 64 at different final

concentrations ranging from 25 to 200 microM Binding reaction was left to take place at room temperature

for 1 h 5 microL of 1 gmL in H2O of glycerol was added to each reaction mixture and loaded on a TA (40

mM TrisndashAcetate) 1 agarose gel At the end of the binding reaction 1 L (001 mg) of ethidium

bromide solution is added The gel was run for 25 h in TA buffer at 10 Vcm EDTA was omitted from

the buffers because it competes with DNA in the reaction

81

Chapter 5

5 Complexation with Gd(III) of carboxyethyl cyclopeptoids as possible contrast agents in MRI

51 Introduction

Tomography of magnetic resonance (Magnetic Resonance Imaging MRI) has gained great

importance in the last three decades in medicinal diagnostics as an imaging technique with a superior

spatial resolution and contrast The most important advantage of MRI over the competing radio-

diagnostic methods such as X-Ray Computer Tomography (CT) Single-Photon Emission Computed

Tomography (SPECT) or Positron Emission Tomography (PET) is definitely the absence of harmful

high-energy radiations Moreover MRI often represents the only reliable diagnostic method for

egcranial abnormalities or multiple sclerosis123

In the course of time it was found that in some

examinations of eg the gastrointestinal tract or cerebral area the information obtained from a simple

MRI image might not be sufficient In these cases the administration of a suitable contrast enhancing

agent (CA) proved to be extremely useful Quite soon it was verified that the most capable class of CAs

could be some compounds containing paramagnetic metal ions

These drugs would be administered to a patient in order to (1) improve the image contrast between

normal and diseased tissue andor (2) indicate the status of organ function or blood flow124

The image

intensity in 1H NMR imaging largely composed of the NMR signal of water protons depend on the

nuclear relaxation times Complexes of paramagnetic transition and lanthanide ions which can decrease

the relaxation times of nearby nuclei via dipolar interactions have received attention as potential

contrast agents Paramagnetic contrast agents are an integral part of this trend they are unique among

diagnostic agents In tissue these agents are not visualized directly on the NMR image but are detected

indirectly by virtue of changes in proton relaxation behavior Moreover the expansion of these agents

offers interesting challenges for investigators in the chemical physical and biological sciences1 These

comprise the design and synthesis of stable nontoxic and tissue-specific metal complexes and the

quantitative understanding of their effect on nuclear relaxation behavior in solution and in tissue

Physical principles of MRI rely on the monitoring of the different distribution and properties of water in

the examined tissue and also on a spatial variation of its proton longitudinal (T1) and transversal (T2)

magnetic relaxation times125

All CAs can be divided (according to the site of action) into extracellular

organ-specific and blood pool agents Historically the chemistry of the T1-CAs has been explored more

extensively as the T1 relaxation time of diamagnetic water solutions is typically five-times longer than T2

and consequently easier to be shortened1 From the chemical point of view T1-CAs are complexes of

paramagnetic metal ions such as Fe(III) Mn(II) or Gd(III) with suitable organic ligands

Gadolinium (III) is an optimal relaxation agents for its high paramagnetism (seven unpaired

electrons) and for its properties in term of electronic relaxation126

The presence of paramagnetic Gd

123 P Hermann J Kotek V Kubigravecek and I Lukes Dalton Trans 2008 3027ndash3047 124 Randall E Lauffer Chem Rev 1987 87 901-927 125

The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging ed A E Merbach and E

Tograveth John Wiley amp Sons Chichester (England) 2001 126 S Aime M Botta M Fasano and E Terreno Chemical Society Reviews 1998 27 19-29

82

(III) complexes causes a dramatic enhancement of the water proton relaxation rates and then allows to

add physiological information to the impressive anatomical resolution commonly obtained in the

uncontrasted images

Other general necessities of contrast agent for MRI are low toxicity rapid excretion after

administration good water solubility and low osmotic potential of the solutions clinically used

However the main problem of the medical utilizations of heavy metal ions like the Gd(III) ion is a

significant toxicity of their ―free (aqua-ion) form Thus for clinical use of gadolinium(III) it must be

bound in a complex of high stability and even more importantly it must show a long term resistance to

a transmetallationtranschelation loss of the Gd(III) ion High chelate stability is essential for lanthanide

complexes in medicine because lanthanide ions have known toxicity via their interaction with Ca(II)

binding sites So the preferred metal complexes in addition to showing high thermodynamic (and

possibly kinetic) stability should present at least one water molecule in their inner coordination sphere

in rapid exchange with the bulk solvent in order to affect strongly the relaxation of all solvent protons

The Gd (III) chelate efficiency is commonly evaluated in vitro by the measure of its relaxivity (r1)

that for commercial contrast agents as Magnevist (DTPA 118) Doratem (DOTA 119) Prohance (HP-

DO3A 120) and Omniscan (DTPA-BMA 121) (figure 51) is around 34-35 mM-1

s-1

(20 MHz and

39degC)2

Figure 51 Commercial contrast agents

The observed water proton longitudinal relaxation rate in a solution containing a paramagnetic metal

complex is given by the sum of three contributions (eq 51)2-127

where R1

w is the water relaxation rate in

the absence of the paramagnetic compound R1pis

represents the contribution due to exchange of water

molecules from the inner coordination sphere of the metal ion to the bulk water and R1pos

is the

contribution of solvent molecules diffusing in the outer coordination sphere of the paramagnetic center

The overall paramagnetic relaxation enhancement (Ris

1p + Ros

1p) referred to a 1 mm concentration of a

given Gd(III) chelate is called its relaxivity2

The inner sphere contribution is directly proportional to the molar concentration of the complex-Gd

to the number of water molecules coordinated to the paramagnetic center q and inversely proportional

127

a) J A Peters J Huskens and D J Raber Prog NMR Spectrosc 1996 28 283 b) S H Koenig and R D Brown III

Prog NMR Spectrosc 1990 22 487

N N

NNHOOC

HOOC

COOH

(DOTA)

119

NH

N NH

N

COOH

CONHCH3H3CHNOC

HOOC

DTPA-BMA

121

N N

NNHOOC

HOOC

COOH

OH

CH3HP-DO3A

120

DTPA

NH

N NH

N

COOH

COOHHOOC

HOOC

118

83

to the sum of the mean residence lifetime τM of the coordinate water protons and their relaxtion time

T1M (eq 52)

52 Eq )τ(555

][

51 Eq

1

1111

MM

is

p

os

p

is

p

oobs

T

CqR

RRRR

The latter parameter is directly proportional to the sixth power of the distance between the metal

center and the coordinated water protons (r) and depends on the molecular reorientational time τR of the

chelate on the electronic relaxation times TiE (i=1 2) of the unpararied electrons of the metal and on

the applied magnetic field strength itself (eq 53 and 54)

53 Eq τω1

7τ

τω1

3τ1)S(S

r

γγ

4π

μ

15

2

T

12

c2

2

s

c2

2

c1

2

H

c1

6

GdH

2

H

2

s

22

0

1M

54 Eq τ

1

τ

1

τ

1

τ

1

EMRci i

For resume all parameters

q is the number of water molecules coordinated to the metal ion

tM is their mean residence lifetime

T1M is their longitudinal relaxation time

S is the electron spin quantum number

γS and γH are the electron and the proton nuclear magnetogyric ratios

rGdndashH is the distance between the metal ion and the protons of the coordinated water

molecules

ωH and ωS are the proton and electron Larmor frequencies respectively

tR is the reorientational correlation time

ηS1 and ηS2 are the longitudinal and transverse electron spin relaxation times

The dependence of Ris

1p and Ros

1p on magnetic field is very significant because the analysis of the

magnetic field dependence permits the determination of the major parameters characterizing the

relaxivity of Gd (III) chelate

A significant step for the design and the characterization of more efficient contrast agents is

represented by the investigation of the relationships between the chemical structure and the factors

determining the ability to enhance the water protons relaxation rates The overall relaxivity can be

correlated with a set of physico-chemical parameters which characterize the complex structure and

dynamics in solution Those that can be chemically tuned are of primary importance in the ligand

design (figure 52)1

84

Figure 52 Model of Gd(III)-based contrast agent in solution

Therefore considering the importance of the contrast agents based on Gd (III) the cyclopeptoids

complexing ability of sodium and the analogy between ionic ray of sodium (102 Ǻ) and gadolinium

(094 Ǻ) the design and the synthesis of cyclopeptoids as potential Gd(III) chelates were realized

Consequently taking inspiration from the commercial CAs three different cyclopeptoids (figure 53 65

66 and 67) containing polar and soluble side chains were prepared and their complexing ability of Gd

(III) was evaluated in collaboration with Prof S Aime at the University of Torino

NN

OOO

OH

O

HO O

HO

O

OHO

N NN

O OO OMe

NNNO

OO

MeO

NN

OOO

OH

O

OH

O

HO O

HO

O

HO

O

OHO

NN

OOO

O

OH

O

HO

O

OHO

6566

67 Figure 53 Hexacarboxyethyl cyclohexapeptoid 65 Tricarboxyethyl ciclohexapeptoid 66 and

tetracarboxyethyl cyclopeptoid 67

85

52 Lariat ether and click chemistry

Cyclopeptoid 67 reminds a lariat ether Lariat ethers are macrocyclic polyether compounds having

one or more donor-group-bearing sidearms Sidearms can be attached either to carbon (carbon-pivot

lariat ethers) or to nitrogen (nitrogen-pivot lariat ethers) When more than one sidearm is attached the

number of them is designated using standard prefixes and the Latin word bracchium which means arm

A two-armed compound is thus a bibracchial lariat ether (abbreviated as BiBLE) Cations such as

Na+ Ca

2+ and NH

4+ are strongly bound by these ligands

128

We have expanded the lariat ethers concept to cyclopeptoids as well as macrocycles and we have

included molecules having sidearms that contain a donor group These sidearms were incorporated into

the cyclic skeleton with a practical and rapid method the ―click chemistry It consists in a chemistry

tailored to generate substances quickly and reliably by joining small units together Of the reactions

comprising the click universe the ―perfect example is the Huisgen 13-dipolar cycloaddition129

of

alkynes to azides to form 14-disubsituted-123-triazoles (scheme 51) The copper(I)-catalyzed reaction

is mild and very efficient requiring no protecting groups and no purification in many cases130

The

azide and alkyne functional groups are largely inert towards biological molecules and aqueous

environments which allows the use of the Huisgen 13-dipolar cycloaddition in target guided

synthesis131

and activity-based protein profiling The triazole has similarities to the ubiquitous amide

moiety found in nature but unlike amides is not susceptible to cleavage Additionally they are nearly

impossible to oxidize or reduce

N N NR

H

R

N

N N

R

H N

N N

R

H

R

Scheme 51 Huisgen 13-dipolar cycloaddition

Cu(II) salts in presence of ascorbate is used for preparative synthesis of 123-triazoles but it is

problematic in bioconjugation applications However tris[(1-benzyl-1H-123-triazol-4-

yl)methyl]amine has been shown to effectively enhance the copper-catalyzed cycloaddition without

damaging biological scaffolds132

Therefore an important intermediate has been a cyclopeptoid containing two alkyne groups in the

sidearms chains (122 figure 54)

128

GW Gokel K A Arnold M Delgado L Echeverria V J Gatto D A Gustowski J

Hernandez A Kaifer S R Miller and L Echegoyen Pure amp Appl Chem 1988 60 461-465 129

For recent reviews see (a) Kolb H C Sharpless K B Drug Discovery Today 2003 8 1128

(b)Kolb H C et al Angew Chem Int Ed 2001 40 2004 130

(a) Rostovtsev V V et al Angew Chem Int Ed2002 41 2596 (b) Tornoslashe C W et al J Org

Chem 2002 67 3057 131

(a) Manetsch R et al J Am Chem Soc2004 126 12809 (b) Lewis W G et alAngew Chem

Int Ed 2002 41 1053 132

Zhang L et al J Am Chem Soc 2005127 15998

86

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

122

Figure 54 Cyclopeptoid intermediate

531 Synthesis

Initially the synthesis of the linear precursors was accomplished through solid-phase mixed

approach (―submonomer and monomerlsquolsquo approach) consisting in an alternate attachment of the N-

fluorenylmethoxycarbonylNlsquo-carboxymethyl-β-alanine (126 scheme 52) and a two step construction

of monomers remnant added to the resin in standard conditions

O

Br -Cl+H3N O

O

OHN O

O

DIPEA DMF

18 h rt

O

Cl

O Fmoc-Cl =

1) LiOH H2O14-Dioxane 0degC 1h

2) Fmoc-ClNaHCO318 h

HO

O

N O

O

Fmoc

123 124125

DIPEA = N

126

Scheme 52 Synthesis of monomer N-fluorenylmethoxycarbonylNlsquo- carboxymethyl-β-alanine

DIC and HATU-induced couplings and chemoselective deprotections yielded the required oligomers

127 128 and 129 (figure 55)

HON

O

O Ot-Bu

H

6127

HON

O

O Ot-Bu

3N

O

H

OMe128

HON

O

O Ot-Bu

2

N

O

N

O

H

Ot-BuO

129

Figure 55 Linear cyclopeptoids

87

All linear compounds were successfully synthesized as established by mass spectrometry with

isolated crude yields between 62 and 70 and purities greater than 90 by HPLC Head-to-tail

macrocyclizations of the linear protect N-substituted glycines were realized in the presence of HATU in

DMF according to our precedent results (figure 56)133

HATU DIPEA

DMF 654

NN

N

NN

OO

O

Ot-Bu

O

Ot-Bu

O

t-BuO O

t-BuO

O

t-BuO

O

Ot-BuO

HON

O

O Ot-Bu

H

6

127

130

HON

O

O Ot-Bu

3

N

O

H

OMe128

NN

N

NN

OO

O

Ot-Bu

O

t-BuO

O

Ot-BuO

HATU DIPEA

DMF 82

131

HON

O

O Ot-Bu

2

N

O

N

O

H

Ot-BuO

129

HATU DIPEA

DMF 71

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

122

Figure 56 Synthesis of Protected cyclopeptoids

The Boc-protected cyclic precursors (130 and 131) were deprotected with trifluoroacetic acid (TFA)

to afford 65 and 66 While Boc-protected cyclic 122 was reacted with azide 132 through click

chemistry to afford protected cyclic 133 (figure 57)

133 Maulucci I Izzo I Bifulco G Aliberti A De Cola C Comegna D Gaeta C Napolitani A Pizza C

Tedesco C Flot D De Riccardis F Chem Commun 2008 3927-3929

88

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

CuSO4 5H2O

sodium ascorbate

H2OCH3OH

N NN

O O

O OMe

NNNO

OO

MeO

NO

OO

NN OMe2

53

122

133

132

Figure 57 Click chemistry reaction

Finally cyclopeptoid 133 was deprotected in presence of trifluoroacetic acid (TFA) in CH2Cl2 to

afford 67

532 Stability evaluation of 65 and 66 as metal complexes

The degree of toxicity of a metal chelate is related to its in vivo degree of dissociation before

excretion

The relaxation rate of the cyclopeptoid solutions (~2 mM) was evaluated Cyclopeptoids 65 and 66

were titrated with a solution of GdCl3 (112 mM) to establish their purity (pH=7 25degC and 20 MHz

figure 57)

Figure 58 Titration of cyclopeptoids 65 and 66 with GdCl3

89

The relaxation rate linearly increased adding Gd(III) Its pendence represents the relaxivity (R1p) of

complex Gd-65 and Gd-66 When Gd(III) was added in excess the line changes its pendence and

followes the relaxivity increase typical of Gd aquaion (R1p= 13 mM-1s-1)

R1oss = R1W + r1p[Gd-CP] Eq 55

CP = cyclopeptoid

R1W is the water relaxation rate in absence of the paramagnetic compound (= 038) Purity was been

of 73 and 56 for 65 and 66 respectively From these data we calculated the complexes relaxivity

which was 315 mM-1

s-1

e 253 mM-1

s-1

for Gd-65 and Gd-66 respectively These values resulted higher

when compared with the commercial contrast agents (~4-5 mM-1

s-1

)

By measuring solvent longitudinal relaxation rates over a wide range of magnetic fields with a field-

cycling spectrometer that rapidly switches magnetic field strength over a range corresponding to proton

Larmor frequencies of 001ndash70 MHz we calculate important relaxivity parameters The data points

represent the so-called nuclear magnetic relaxation dispersion (NMRD) profile that can be adequately

fitted to yield the values of the relaxation parameters (figure 59)

Figure 59 1T1 NMRD profiles for Gd-65 and Gd-66

The efficacy of the CA measured as the ability of its 1 mM solution to increase the longitudinal

relaxation R1 (=1T1) of water protons is called relaxivity and labeled r1 According to the well

established Solomon-Boembergen-Morgan theory and its improvement called generalized SBM134

the

relaxivity parameters (see eq 51-54) were evaluated and reported into table 51

134

E Strandeberg P O Westlund J Magn Reson Ser A 1996 122 179-191

90

Table 51 Parameters determined by SBM theory

2 (s

-2) v (ps) M (s) R (ps) q qass

Gd-65 21times1019

275 1times10-8

280 3 15

Gd-66 28times1019

225 1times10-8

216 3 14

Electronic relaxation parameters (2 e v) were similar for complexes Gd-65 and Gd-66 and

comparable to commercial contrast agents

From the data reported (see q and qass) appears that Gd-65 and Gd-66 complexes coordinate directly

(in the inner sphere see figure 52) 3 water molecules and about 14-15 water molecules in the second

coordination sphere

Finally we have also tested the stability of the complex Gd-65 in solution using EDTA as competitor

(logKEDTA=1735) in a titration (figure 510 increased EDTA amounts into Gd-65 solution 0918 mM

pH 7) Being stability constant of Gd-EDTA complex known and once knew Gd-65 relaxivity it was

possible to fit these experimental data and obtain stability constant of the examined complex

Figure 510 Tritation profile of Gd-65 with EDTA

The stability constant was determinated to be logKGd-65 = 1595 resulting too low for possible in vivo

applications The stability studies for the complexes Gd-66 and Gd-67 are in progress

541 Synthesis

Compound 125

To a solution of β-alanine hydrochloride 124 (30 g 165 mmol) in DMF dry (5 mL) DIPEA (574

mL 330 mmol) and ethyl bromoacetate (093 mL 825 mmol) were added The reaction mixture was

stirred overnight concentrated in vacuo dissolved in CH2Cl2 (20 mL) and washed with brine solution

The aqueous layer was extracted with CH2Cl2 (three times) The combined organic phases were dried

over MgSO4 filtered and the solvent evaporated in vacuo to give a crude material 125 (30 g 100

yellow oil) which was used in the next step without purification δH (30010 MHz CDCl3) 131 (3H t J

91

90 Hz CH3CH2) 147 (9H s C(CH3)3) 274 (2H t J 60 Hz NHCH2CH2CO) 309 (2H t J 90 Hz

NHCH2CH2CO) 365 (2H s CH2NHCH2CH2CO) 426 (2H q J 150 Hz) δC (7550 MHz CDCl3)

1401 2797 3350 4430 4914 6041 8144 16888 17077 mz (ES) 232 (MH+) (HRES) MH+

Compound 126

To a solution of 125 (30 g 130 mmol) in 14-dioxane (30 mL) at 0degC LiOHbullH2O (0587 g 140

mmol) in H2O (30 mL) was added After two hours NaHCO3 (142 g 99 mmol) and Fmoc-Cl (433 g

99 mmol) were added The reaction mixture was stirred overnight Subsequently KHSO4 (until pH= 3)

was added and concentrated in vacuo dissolved in CH2Cl2 (20 mL) The aqueous layer was extracted

with CH2Cl2 (three times) The combined organic phases were dried over MgSO4 filtered and the

solvent evaporated in vacuo to give a crude material (58 g g yellow oil) which was purified by flash

chromatography (CH2Cl2CH3OH from 1000 to 8020 01 ACOH) to give 126 (50 mg 30) δH

(30010 MHz CDCl3 mixture of rotamers) 144 (9H s C(CH3)3) 232 (12H t J 64 Hz

NHCH2CH2CO rot a) 258 (08H t J 60 Hz NHCH2CH2CO rot b) 345 (12H t J 63 Hz

NHCH2CH2CO rot a) 359 (08H t J 59 Hz NHCH2CH2CO rot b) 405 (08H s

CH2NHCH2CH2CO rot b) 413 (12H s CH2NHCH2CH2CO rot a) 420 (04H t J 60 Hz

CH2CHFmoc rot b) 428 (06H t J 60 Hz CH2CHFmoc rot a) 445 (12H d J 63 Hz

CH2CHFmoc rot b) 455 (08H d J 60 Hz CH2CHFmoc rot a) 719-744 (4H m Ar (Fmoc))

753 (08H d J 73 Hz Ar (Fmoc) rot b) 759 (12H d J 73 Hz Ar (Fmoc) rot a) 773 (08H t J

73 Hz Ar (Fmoc) rot b) 778 (12H t J 73 Hz Ar (Fmoc) rot a) δC (6289 MHz CDCl3 mixture

of rotamers) 2790 3442 3460 4438 4509 4703 (times 2) 4971 4999 6759 8087 11984 12470

(times 2) 12516 (times 2) 12691 12700 12759 (times 2) 12808 12889 14119 14362 15563 15624

17111 17161 17488 17504 mz (ES) 454 (MH+) (HRES) MH

+ found 454229 C26H32NO6

+

requires 454223

542 Linear compounds 127 128 and 129

Linear peptoids 127 128 and 129 were synthesized by alternating submonomer and monomer solid-

phase method using standard manual Fmoc solid-phase peptide synthesis protocols Typically 020 g of

2-chlorotrityl chloride resin (Fluka 2α-dichlorobenzhydryl-polystyrene crosslinked with 1 DVB

100-200 mesh 120 mmolg) was swelled in dry DCM (2 mL) for 45 min and washed twice in dry

DCM (2 mL) To the resin monomer 126 (68 mg 016 mmol) and DIPEA (011 mL 064 mmol) in dry

DCM (2 mL) were added and putted on a shaker platform for 1h and 15 min at room temperature

washes with dry DCM (3 times 2mL) and then with DMF (3 times 2 mL) followed The resin was capped with a

solution of DCMCH3OHDIPEA The Fmoc group was deprotected with 20 piperidineDMF (vv 3

mL) on a shaker platform for 3 min and 7 min respectively followed by extensive washes with DMF (3

times 3 mL) DCM (3 times 3 mL) and DMF (3 times 3 mL) Subsequently for compound 130 the resin was

incubated with a solution of monomers 126 (064 mmol) HATU (024 g 062 mmol) DIPEA (220 μL

128 mmol) in dry DMF (2 mL) on a shaker platform for 1 h followed by extensive washes with DMF

(3 times 2 mL) DCM (3 times 2 mL) and DMF (3 times 2 mL) For compounds 128 and 129 instead

bromoacetylation reactions were accomplished by reacting the oligomer with a solution of bromoacetic

acid (690 mg 48 mmol) and DIC (817 μL 528 mmol) in DMF (4 mL) on a shaker platform 40 min at

92

room temperature Subsequent specific amine was added (methoxyethyl amine 017 mL 20 mmol or

propargyl amine 015 mL 24 mmol)

Chloranil test was performed and once the coupling was complete the Fmoc group was deprotected

with 20 piperidineDMF (vv 3 mL) on a shaker platform for 3 min and 7 min respectively followed

by extensive washes with DMF (3 times 3 mL) DCM (3 times 3 mL) and DMF (3 times 3 mL) The yields of

loading step and of the following coupling steps were evaluated interpolating the absorptions of

dibenzofulvene-piperidine adduct (λmax = 301 ε = 7800 M-1 cm-1) obtained in Fmoc deprotection

step (the average coupling yield was 63-70)

The synthesis proceeded until the desired oligomer length was obtained The oligomer-resin was

cleaved in 4 mL of 20 HFIP in DCM (vv) The cleavage was performed on a shaker platform for 30

min at room temperature the resin was then filtered away The resin was treated again with 4 mL of 20

HFIP in DCM (vv) for 5 min washed twice with DCM (3 mL) filtered away and the combined filtrates

were concentrated in vacuo The final products were dissolved in 50 ACN in HPLC grade water and

analysed by RP-HPLC and ESI mass spectrometry

Compound 127 tR 181 min mz (ES) 1129 (MH+) (HRES) MH

+ found 1129 6500

C54H93N6O19+ requires 11296425 80

Compound 128 tR 151 min m z (ES) 919 (MH+) (HRES) MH

+ found 9195248

C42H75N6O16+ requires 9195240 75

+ found 9495138

C43H73N6O15+ requires 9495134 85

Linear 127 (0110 g 0097 mmol) was dissolved in DMF dry (8 mL) and the mixture was

added drop-wise by syringe pump in 2 h to a stirred solution of HATU (015 g 039 mmol) and DIPEA

(010 mL 060 mmol) in dry DMF (25 mL) at room temperature in anhydrous atmosphere

added drop-wise by syringe pump in 2 h to a stirred solution of HATU (0093 g 0244 mmol) and

DIPEA (0065 mL 038 mmol) in dry DMF (15 mL) at room temperature in anhydrous atmosphere

93

39 mm x 300 mm]) and ESI mass spectrometry (zoom scan technique)

Compound 130 δH (30000 MHz CDCl3 mixture of conformers) 144 (54H br s C(CH3)3)

254-358 (24H m CH2CH2COOt-Bu) 394-450 (12H m CH2 intranular) δC (6289 MHz CDCl3

mixture of conformers) 2799 2963 3320 3372 3402 3415 3474 4380 4433 4458 4504

4531 4555 4572 4594 4676 4756 4774 4858 4896 4977 5004 5037 5102 5277 5323

5340 8058 8141 16765 16776 16793 16819 16832 16856 16886 16890 16902 16915

16928 16938 16951 16993 17027 17038 17062 17069 17078 17091 17096 17114

17134 17139 17158 17167 17174 17187 65 mz (ES) 1111 (MH+) (HRES) MH

+ found

11116395 C54H91N6O18+

Compound 130 with sodium picrate δH (400 MHz CD3CNCDCl3 91 25 degC soluzione 40

mM) 144 (54H s C(CH3)3) 256 (12H t J 80 Hz CH2CH2COOt-Bu) 347 (6H m CHHCH2COOt-

Bu) 361 (6H m CHHCH2COOt-Bu) 392 (6H d J 160 Hz -OCCHHN pseudoequatorial) 461 (6H

d J 200 Hz -OCCHHN pseudoaxial) 877 (3H s Picrate) δC (100 MHz CD3CNCDCl3 91 25 degC

solution 40 mM) 2846 3478 4550 5020 8227 12724 12822 14310 17015 17173

Compound 131 δH (40010 MHz CDCl3 mixture of conformers) 143-146 (54H br s

C(CH3)3) 325-360 (33H m CH2CH2COOt-Bu e CH2CH2OCH3) 394-465 (12H m CH2 intranular)

δC (6289 MHz CDCl3 mixture of conformers) 2913 2933 2956 3501 3541 4517 4635 4720

4962 4992 5061 5397 5993 6026 7280 8195 8110 8269 11401 11800 16061 16072

17001 17075 17121 17145 17190 17219 17245 17288 17310 82 mz (ES) 901 (MH+)

Compound 131 with sodium picrate δH (400 MHz CD3CNCDCl3 91 solution 40 mM)

144 (27H s C(CH3)3) 256 (6H t J 80 Hz CH2CH2COOt-Bu) 332 (9H s CH2CH2OCH3) 347-

370 (18H m CHHCH2COOt-Bu e CHHCH2COOt-Bu e CH2CH2OCH3) 381 (3H d J 167 Hz -

OCCHHN pseudoequatorial) 389 (3H d J 170 Hz -OCCHHN pseudoequatorial) 464 (3H d J 167

Hz -OCCHHN pseudoaxial) 468 (3H d J 166 Hz -OCCHHN pseudoaxial) 874 (3H s Picrate)

C(CH3)3) 215-280 (10H m CH2CH2COOt-Bu e CH2CCH) 350-465 (24H m CH2CCH CH2

intranular e CH2CH2COOt-Bu) (6289 MHz CDCl3 mixture of conformers) 2787 2950 3359 3416

3653 3671 3698 4378 4402 4421 4437 4509 4687 4755 4767 4780 4866 4903 4925

4991 5016 5053 5091 5297 5329 7236 7250 7286 8056 8127 8136 15786 15863

16720 16805 16851 16889 17026 17100 17151 17152 71 mz (ES) 931 (MH+) (HRES)

MH+ found 9315029 C46H71N6O14

94

544 Synthesis of 133 by click chemistry

Compound 122 (0010 g 0011 mmol) was dissolved in CH3OH (55 μL) and compound 132 (0015 g

0066 mmol) was added and the reaction was stirred for 15 minutes Subsequent a solution of CuSO4

penta hydrate (0001 g 00044 mmol) and sodium ascorbate (00043 g 0022 mmol) in water (110 μL)

was slowly added The reaction was stirred overnight After at the solution H2O (03 ml) was added and

the single mixture was extracted with CH2Cl2 (2 x 20 mL) and the combined organic phases were

washed with water (12 mL) dried over anhydrous Na2SO4 filtered and concentrated in vacuo The

crude residue 133 was purified by HPLC on a C18 reversed-phase preparative column conditions 20-

100 B in 40 min [A 01 TFA in water B 01 TFA in acetonitrile] flow 20 mLmin 220 nm The

samples were dried in a falcon tube under low pressure (53 ) δH (40010 MHz CDCl3 mixture of

conformers) 142 (36H br s C(CH3)3) 254 (8H m CH2CH2COOt-Bu) 330-505 (62H m

CH2CH2COOt-Bu NCH2C=CH CH2 etilen-glicole CH2 intranulari e OCH3) 790 (2H m C=CH) δC

(10003 MHz CDCl3 mixture of conformers) 1402 2256 2804 2962 3193 3299 3373 4227

4301 4448 4501 4564 4838 4891 4944 5060 5334 5892 6915 6999 7052 7189 8054

8069 8080 8092 8136 11387 11640 12416 12435 12446 12465 12474 12532 12547

14221 15891 15941 15952 16872 16954 17140 17055 17100 17120 17131 mz (ES) 1397

(MH+) (HRES) MH

+ found 13977780 C64H109N12O22

Protected cyclopeptoids 130 (0058 g 0052mmol) 131 (0018 g 0020 mmol) and 122 (0010 g

00072 mmol) were dissolved in a mixture of m-cresol and TFA (19 13 mL for 130 05 mL for 131

018 mL for 122) and the reaction was stirred for one hour After products were precipitated in cold

Ethyl Ether (20 mL) and centrifuged Solids were recuperated with a quantitative yield

254-358 (24H m CH2CH2COOt-Bu) 394-450 (12H m CH2 intranular) δc (6289 MHz CDCl3

4531 4555 4572 4594 4676 4756 4774 4858 4896 4977 5004 5037 5102 5277 5323

5340 8058 8141 16765 16776 16793 16819 16832 16856 16886 16890 16902 16915

16928 16938 16951 16993 17027 17038 17062 17069 17078 17091 17096 17114

17134 17139 17158 17167 17174 17187 mz (ES) 775 (MH+) (HRES) MH

+ found 7752638

C30H43N6O18+

Compound 65 with sodium picrate δH (40010 MHz CD3CND2OMeOD=211 spettro

151 complesso) 260 (12H m CH2CH2COOH) 340 (6H m CHHCH2COOH overlapped with

water signal) 354 (6H m CHHCH2COOH) 385 (6H d J 165 Hz -OCCHHN pseudoequatorial)

472 (6H d J 171 Hz -OCCHHN pseudoaxial) 868 (3H s Picrate)

Compound 66 δH (30000 MHz CDCl3 mixture of rotamers) 324-480 (45H complex

signal CH2CH2OCH3 CH2CH2COOH CH2 intranular) δc (6289 MHz CD3CNMeOD=91 mixture of

rotamers) 1459 3143 3214 3258 4359 4400 4460 4504 4574 4931 4969 5004 5757

5777 5785 5802 5829 6551 6942 6957 7011 7021 7035 16870 16891 16898 16934

16945 16957 16987 16997 17035 17083 17110 17160 17182 17275 17298 17318

95

17330 mz (ES) 733 (MH+) (HRES) MH

+ found 7333259 C30H49N6O15

tR 843 min

Compound 66 with sodium picrate δH (30000 MHz CDCl3 mixture of rotamers) 261 (6H

br s CH2CH2COOH overlapped with water signal) 330 (9H s CH2CH2OCH3) 350-360 (18H m

CHHCH2COOH CHHCH2COOH e CH2CH2OCH3) 377 (3H d J 210 Hz -OCCHHN

pseudoequatorial) 384 (3H d J 210 Hz -OCCHHN pseudoequatorial) 464 (3H d J 150 Hz -

OCCHHN pseudoaxial) 483 (3H d J 150 Hz -OCCHHN pseudoaxial) 868 (3H s picrate)

Compound 67 δH (40010 MHz CDCl3 mixture of rotamers) 299-307 (8H m

CH2CH2COOt-Bu) 370 (6H s OCH3) 380-550 (56H m CH2CH2COOt-Bu NCH2C=CH CH2

ethyleneglicol CH2 intranular) 790 (2H m C=CH) HPLC tR 1013 min

96

Chapter 6

6 Cyclopeptoids as mimetic of natural defensins

61 Introduction

The efficacy of antimicrobial host defense in animals can be attributed to the ability of the immune

system to recognize and neutralize microbial invaders quickly and specifically It is evident that innate

immunity is fundamental in the recognition of microbes by the naive host135

After the recognition step

an acute antimicrobial response is generated by the recruitment of inflammatory leukocytes or the

production of antimicrobial substances by affected epithelia In both cases the hostlsquos cellular response

includes the synthesis andor mobilization of antimicrobial peptides that are capable of directly killing a

variety of pathogens136

For mammals there are two main genetic categories for antimicrobial peptides

cathelicidins and defensins2

Defensins are small cationic peptides that form an important part of the innate immune system

Defensins are a family of evolutionarily related vertebrate antimicrobial peptides with a characteristic β-

sheet-rich fold and a framework of six disulphide-linked cysteines3 Hexamers of defensins create

voltage-dependent ion channels in the target cell membrane causing permeabilization and ultimately

cell death137

Three defensin subfamilies have been identified in mammals α-defensins β-defensins and

the cyclic θ-defensins (figure 61)138

α-defensin

135 Hoffmann J A Kafatos F C Janeway C A Ezekowitz R A Science 1999 284 1313-1318 136 Selsted M E and Ouelette A J Nature immunol 2005 6 551-557 137 a) Kagan BL Selsted ME Ganz T Lehrer RI Proc Natl Acad Sci USA 1990 87 210ndash214 b)

Wimley WC Selsted ME White SH Protein Sci 1994 3 1362ndash1373 138 Ganz T Science 1999 286 420ndash421

97

β-defensin

θ-defensin

Figure 61 Defensins profiles

Defensins show broad anti-bacterial activity139

as well as anti-HIV properties140

The anti-HIV-1

activity of α-defensins was recently shown to consist of a direct effect on the virus combined with a

serum-dependent effect on infected cells141

Defensins are constitutively produced by neutrophils142

or

produced in the Paneth cells of the small intestine

Given that no gene for θ-defensins has been discovered it is thought that θ-defensins is a proteolytic

product of one or both of α-defensins and β-defensins α-defensins and β-defensins are active against

Candida albicans and are chemotactic for T-cells whereas θ-defensins is not143

α-Defensins and β-

defensins have recently been observed to be more potent than θ-defensins against the Gram negative

bacteria Enterobacter aerogenes and Escherichia coli as well as the Gram positive Staphylococcus

aureus and Bacillus cereus9 Considering that peptidomimetics are much stable and better performing

than peptides in vivo we have supposed that peptoidslsquo backbone could mimic natural defensins For this

reason we have synthesized some peptoids with sulphide side chains (figure 62 block I II III and IV)

and explored the conditions for disulfide bond formation

139 a) Ghosh D Porter E Shen B Lee SK Wilk D Drazba J Yadav SP Crabb JW Ganz T Bevins

CL Nat Immunol 2002 3 583ndash590b) Salzman NH Ghosh D Huttner KM Paterson Y Bevins CL

Nature 2003 422 522ndash526 140 a) Zhang L Yu W He T Yu J Caffrey RE Dalmasso EA Fu S Pham T Mei J Ho JJ Science

2002 298 995ndash1000 b) 7 Zhang L Lopez P He T Yu W Ho DD Science 2004 303 467 141 Chang TL Vargas JJ Del Portillo A Klotman ME J Clin Invest 2005 115 765ndash773 142 Yount NY Wang MS Yuan J Banaiee N Ouellette AJ Selsted ME J Immunol 1995 155 4476ndash

4484 143 Lehrer RI Ganz T Szklarek D Selsted ME J Clin Invest 1988 81 1829ndash1835

98

HO

NN

NNH

OO

O

STr STr

NHBoc NHBoc68

N

NN

N

NNO

O

OO

O

NHBoc

SH

BocHN

HS

69N

NN

N

NNO

O

OO

O

NHBoc

S

BocHN

S

70

N

NN

N

O O

O

OOO

O

SH

HS

N

NN

N

O O

O

OO

O

S

72 73

NN

OO

O

STr

71

N

O

NH

STr

HO

99

OHNN

N

NN

OO

O

OO

O

TrS

NOO

N

NN

NNH

OO

O

TrS

74N

N

N N

NN

N

NO

O

O O O

OOO

O

NO

SH

HS

N

N N

NN

N

NO

O

O O O

OOO

O

NO

S

75

76

OH

NN

N

OO

O

S

NO

O

N

NH

O

S

77

HO

NN

OO O

OO

OTrS

78

NOO

N

HN

O

O STr

N

N N

NN

N

NO

O

O O O

OOO

O

NO

HS

79

SH

N

N N

NN

N

NO

O

O O O

OOO

O

NO

S

80

S

HO

NN

OO O

OO

O

S

NOO

N

HN

O

OS

81

Figure 62 block IV Structures of dodecameric linear diprolinate (78) and corresponding cyclic 79

80 and 81

100

Disulfide bonds play an important role in the folding and stability of many biologically important

peptides and proteins Despite extensive research the controlled formation of intramolecular disulfide

bridges still remains one of the main challenges in the field of peptide chemistry144

The disulfide bond formation in a peptide is normally carried out using two main approaches

(i) while the peptide is still anchored on the resin

(ii) after the cleavage of the linear peptide from the solid support

Solution phase cyclization is commonly carried out using air oxidation andor mild basic

conditions10

Conventional methods in solution usually involve high dilution of peptides to avoid

intermolecular disulfide bridge formation On the other hand cyclization of linear peptides on the solid

support where pseudodilution is at work represents an important strategy for intramolecular disulfide

bond formation145

Several methods for disulfide bond formation were evaluated Among them a recently reported on-

bead method was investigated and finally modified to improve the yields of cyclopeptoids synthesis

10

621 Synthesis

In order to explore the possibility to form disulfide bonds in cyclic peptoids we had to preliminarly

synthesized the linear peptoids 68 71 74 and 78 (Figure XXX)

To this aim we constructed the amine submonomer N-t-Boc-14-diaminobutane 134146

and the

amine submonomer S-tritylaminoethanethiol 137147

as reported in scheme 61

NH2

CH3OH Et3N

H2NNH

O

134 135O O O

O O

(Boc)2O

NH2

SHH2N

S(Ph)3COH

TFA rt quant

136 137

Scheme 61 N-Boc protection and S-trityl protection

The synthesis of the liner peptoids was carried out on solid-phase (2-chlorotrityl resin) using the

The identity of compounds 68 71 74 and 78 was established by mass

spectrometry with isolated crudes yield between 60 and 100 and purity greater than 90 by

144 Galanis A S Albericio F Groslashtli M Peptide Science 2008 92 23-34 145 a) Albericio F Hammer R P Garcigravea-Echeverrıigravea C Molins M A Chang J L Munson M C Pons

M Giralt E Barany G Int J Pept Protein Res 1991 37 402ndash413 b) Annis I Chen L Barany G J Am Chem

Soc 1998 120 7226ndash7238 146 Krapcho A P Kuell C S Synth Commun 1990 20 2559ndash2564 147 Kocienski P J Protecting Group 3rd ed Georg Thieme Verlag Stuttgart 2005 148 Zuckermann R N Kerr J M Kent B H Moos W H J Am Chem Soc 1992 114 10646

101

HPLCMS analysis149

Head-to-tail macrocyclization of the linear N-substituted glycines was performed in the presence of

HATU in DMF and afforded protected cyclopeptoids 138 139 140 and 141 (figure 63)

N

NN

N

O O

O

OO

O

STr

TrS

139

N

NN

N

NNO

O

NHBoc

STr

BocHN

TrS

138

N

N N

NN

N

O

O O O

OO

O

N

O

NO

STr

TrS

140

N

N N

N

O

O O O

OO

O

N

O

NO

TrS

141 STr

Figure 63 Protected cyclopeptoids 138 139 140 and 141

The subsequent step was the detritylationoxidation reactions Triphenylmethyl (trityl) is a common

S-protecting group150

Typical ways for detritylation usually employ acidic conditions either with protic

acid151

(eg trifluoroacetic acid) or Lewis acid152

(eg AlBr3) Oxidative protocols have been recently

149 Analytical HPLC analyses were performed on a Jasco PU-2089 quaternary gradient pump equipped with an

MD-2010 plus adsorbance detector using C18 (Waters Bondapak 10 μm 125Aring 39 times 300 mm) reversed phase

columns 150 For comprehensive reviews on protecting groups see (a) Greene T W Wuts P G M Protective Groups in

Organic Synthesis 2nd ed Wiley New York 1991 (b) Kocienski P J Protecting Group 3rd ed Georg Thieme

Verlag Stuttgart 2005 151 (a) Zervas L Photaki I J Am Chem Soc 1962 84 3887 (b) Photaki I Taylor-Papadimitriou J

Sakarellos C Mazarakis P Zervas L J Chem Soc C 1970 2683 (c) Hiskey R G Mizoguchi T Igeta H J

Org Chem 1966 31 1188 152 Tarbell D S Harnish D P J Am Chem Soc 1952 74 1862

102

developed for the deprotection of trityl thioethers153

Among them iodinolysis154

in a protic solvent

such as methanol is also used16

Cyclopeptoids 138 139 140 and 141 and linear peptoids 74 and 78

were thus exposed to various deprotectionoxidation protocols All reactions tested are reported in Table

61

Table 61 Survey of the detritylationoxidation reactions

One of the detritylation methods used (with subsequent disulfide bond formation entry 1) was

proposed by Wang et al155

(figure 64) This method provides the use of a catalyst such as CuCl into an

aquose solvent and it gives cleavage and oxidation of S-triphenylmethyl thioether

Figure 64 CuCl-catalyzed cleavage and oxidation of S-triphenylmethyl thioether

153 Gregg D C Hazelton K McKeon T F Jr J Org Chem 1953 18 36 b) Gregg D C Blood C A Jr

J Org Chem 1951 16 1255 c) Schreiber K C Fernandez V P J Org Chem 1961 26 2478 d) Kamber B

Rittel W Helv Chim Acta 1968 51 2061e) Li K W Wu J Xing W N Simon J A J Am Chem Soc 1996

118 7237 154

K W Li J Wu W Xing J A Simon J Am Chem Soc 1996 118 7236-7238

155 Ma M Zhang X Peng L and Wang J Tetrahedron Letters 2007 48 1095ndash1097

Compound Entries Reactives Solvent Results

138

1

2

3

4

CuCl (40) H2O20

TFA H2O Et3SiH

(925525)17

I2 (5 eq)16

DMSO (5) DIPEA19

CH2Cl2

TFA

AcOHH2O (41)

CH3CN

-

139

5

6

7

8

9

TFA H2O Et3SiH

(925525)17

DMSO (5) DIPEA19

DMSO (5) DBU19

K2CO3 (02 M)

I2 (5 eq)154

TFA

CH3CN

THF

CH3OH

-

140

9 I2 (5 eq)154

CH3OH gt70

141

9 I2 (5 eq)154

CH3OH gt70

74

9 I2 (5 eq)154

CH3OH gt70

78

9 I2 (5 eq)154

CH3OH gt70

103

For compound 138 Wanglsquos method was applied but it was not able to induce the sulfide bond

formation Probably the reaction was condizioned by acquose solvent and by a constrain conformation

of cyclohexapeptoid 138 In fact the same result was obtained using 1 TFA in the presence of 5

triethylsilane (TIS17

entry 2 table 61) in the case of iodinolysis in acetic acidwater and in the

presence of DMSO (entries 3 and 4)

One of the reasons hampering the closure of the disulfide bond in compound 138 could have been

the distance between the two-disulfide terminals For this reason a larger cyclooctapeptoid 139 has been

synthesized and similar reactions were performed on the cyclic octamer Unfortunately reactions

carried out on cyclo 139 were inefficient perhaps for the same reasons seen for the cyclo hexameric

138 To overcome this disvantage cyclo dodecamers 140 and 141 were synthesized Compound 141

containing two prolines units in order to induce folding156

of the macrocycle and bring the thiol groups

closer

Therefore dodecameric linears 74 and 78 and cyclics 140 and 141 were subjectd to an iodinolysis

reaction reported by Simon154

et al This reaction provided the use of methanol such as a protic solvent

and iodine for cleavage and oxidation By HPLC and high-resolution MS analysis compound 76 77 80

and 81 were observed with good yelds (gt70)

63 Conclusions

Differents cyclopeptoids with thiol groups were synthesized with standard protocol of synthesis on

solid phase and macrocyclization Many proof of oxdidation were performed but only iodinolysis

reaction were efficient to obtain desidered compound

641 Synthesis

Compound 135

Di-tert-butyl dicarbonate (04 eq 10 g 0046 mmol) was added to 14-diaminobutane (10 g 0114

give 135 (051 g 30) as a yellow light oil Rf (98201 CH2Cl2CH3OHNH3 20M solution in ethyl

alcohol) 063 δH (300 MHz CDCl3) 468 (brs 1H NH-Boc) 312 (bq 2H CH2-NH-Boc J = 75

MHz) 271 (t 2H CH2NH2 J =70 MHz) 18 (brs 2H NH2) 135 (s 9H (CH3)3) mz (ES) 189

(MH+) (HRES) MH

+ found 1891600 C9H21N2O2

+ requires 1891598

156 MacArthur M W Thornton J M J Mol Biol 1991 218 397

104

Compound 137

Aminoethanethiol hydrocloride (5 g 0044 mol) was dissolved in 125 mL of TFA

Triphenylcarbinol (115 g 0044 mol) was added to the solution portionwise under stirring at room

temperature until the solution became clear The reaction mixture a dense deeply red liquid was left

aside for 1 h and the poured in 200 mL of water under vigorous stirring The suspension of the white

solid was alkalinized with triethylamine and filtered and the solid washed with water alkaline for TEA

After drying 18 g of the crude solid of 137 slightly impure for TrOH was obtained The compound

was used in the next step without purification yeld100 Rf (98201 CH2Cl2CH3OHNH3 20M

solution in ethyl alcohol) 063 δH (250 MHz CDCl3) 234 (t 2H CH2-STr J=75 Hz) 341 (t 2H

CH2NH2 J =70 MHz) 719-741 (m 15H Ar) mz (ES) 320 (MH+) (HRES) MH

+ found 3201470

C21H22NS+ requires 3201467 δC (100 MHz CDCl3) 319 (CH2-STr) 401 (CH2NH2) 680 (C(Ph)3)

1278-1285-1289 (CH trityl) 1457 (Cq trityl)

642 General procedures for linear oligomers 68 71 74 and 78

Linear peptoid oligomers 68 71 74 and 78 were synthesized using a sub-monomer solid-phase

approach11

(isobutylamine -10 eq- or 135 -10 eq- or 137 -10 eq) the mixture was left on a shaker platform for 30

min at room temperature then the resin was washed with DMF (4 x 4 mL) Subsequent

bromoacetylation reactions were accomplished by reacting the aminated oligomer with a solution of

bromoacetic acid in DMF (12 M 53 mL) and 823 μL of DIC for 40 min at room temperature The

filtered resin was washed with DMF (4 x 4 mL) and treated again with the amine in the same conditions

reported above This cycle of reactions was iterated until the target oligomer was obtained (68 71 74

and 78 peptoids) The cleavage was performed by treating twice the resin previously washed with DCM

(6 x 6 mL) with 4 mL of 20 HFIP in DCM (vv) on a shaker platform at room temperature for 30 min

and 5 min respectively The resin was then filtered away and the combined filtrates were concentrated

in vacuo The final products were dissolved in 50 acetonitrile in HPLC grade water and analysed by

RP-HPLC (purity gt95 for all the oligomers conditions 5 to 100 B in 30 min [A 01 TFA in

water B 01 TFA in acetonitrile] flow 10 mLmin 220 nm C18 reversed-phase analytical column

[Waters μBondapak 10 μm 125 Aring 39 mm x 300 mm]) and ESI mass spectrometry The linear

oligomers 68 71 74 and 78 were subjected to the cyclization reaction without further purification

+ found 14197497

C80H107N8O11S2+ requires 14197495 100

+ found 14157912

C82H111N8O9S2+ requires 14157910 100

105

+ found 18681278

C106H155N12O13S2+ requires 18681273 100

+ found 18360650

C104H147N12O13S2+ requires 18360647 100

643 General cyclization reaction (synthesis of 138 139 140 and 141)

A solution of the linear peptoid (68 71 74 and 78) previously co-evaporated three times with

Linear 68 (10 eq 200 mg 0141 mmol) was dissolved in DMF dry (10 mL) and the mixture

was added drop-wise by syringe pump in 2 h to a stirred solution of HATU (40 eq 214 mg 0564

mmol) and DIPEA (62 eq 152 microl 0874 mmol) in dry DMF (37 mL) at room temperature in

All protected cyclic 138 139 140 and 141 were dissolved in 50 acetonitrile in HPLC grade water

and analysed by RP-HPLC (purity gt85 for all the cyclic oligomers conditions 5-100 B in 30 min

[A 01 TFA in water B 01 TFA in acetonitrile] flow 10 mLmin 220 nm C18 reversed-phase

30H H-Ar) 421-248 (m 34H NCH2CH2CH2CH2NHCO(CH3)3 -COCH2N- NCH2CH(CH3)2

CH2CH2STr overlapped) 143 (m 26 H NCH2CH2CH2CH2NHCO(CH3)3) 072-090 (m 12 H

NCH2CH(CH3)2) mz (ES) 1401 (MH+) (HRES) MH

+ found 14017395 C80H105N8O10S2

+ requires

14017390 HPLC tR 250 min

106

490-234 (m 38H NCH2CH(CH3)2 CH2CH2STr -COCH2N- overlapped) 145-070 (m 36H

+ found 13977810 C82H109N8O8S2

+ requires

13977804 HPLC tR 271 min

+ found 18501170 C106H153N12O12S2

+ requires

18501167 HPLC tR 330 min

490-223 (m 70H NCH2CH(CH3)2 CH2CH2STr -COCH2N- CHCH2CH2CH2NCO overlapped)

145-070 (m 48H NCH2CH(CH3)2) mz (ES) 1804 (MH+) (HRES) MH

+ found 18040390

C103H143N12O12S2+ requires 18040384 HPLC tR 291 min

644 General DeprotectionOxidation reactions reported in table 61 (synthesis of 69-70 72-73

75-76-77 and 79-80-81)

General procedure for Entry 1

Compound 68 (30 mg 0021 mmol) was dissolved in CH2Cl2 (10 mL) and to this solution was

successively added CuCl (09 mg 0009 mmol) and H2O (08 mg 0042 mmol) then the reaction bottle

was sealed with a rubber plug The reaction was conducted under ultrasonic irradiation for 3ndash7 h until

detritylation was complete as judged by HPLCMS

General procedures for Entry 2 and 5

Compound 68 (27 mg 0019 mmol) and Compound 139 (20 mg 0014 mmol) was dissolved in a

mixture of TFAH2OEt3SiH (925525) and the reaction was stirred for 1h and 30min After products

were precipitated in cold Ethyl Ether (20 mL) and centrifuged Products recuperated was analyzed by

HPLCMS

General procedure of Entry 3

Compound 68 (5 mg 0007 mmol) was dissolved in 175 mL solution mixture of AcOH-H2O (41)

containing 9 mg of I2 (0035 mmol 5 equivalents) The reaction mixture was stirred for 3 h then

mixture was concentrated in vacuo and analyzed by HPLCMS

General procedure for Entry 4 and 6

Compound 68 (10 mg 0014 mmol) and compound 139 (10 mg 0011 mmol) were dissolved in

about 11-13 mL of CH3CN and 5 of DMSO and then DIPEA (24 μL 014 mmol for 68 and 19 μL

011 mmol for 139) were added The mixtures were stirred for overnight Mixtures were concentrated in

vacuo and analyzed by HPLCMS

107

Compound 139 (15 mg 00016 mmol) was dissolved in about 16 mL of CH3CN and 5 of DMSO

and then DBU (24 μL 0016 mmol) were added The mixture was stirred for overnight Mixture was

concentrated in vacuo and analyzed by HPLCMS

Compound 139 (15 mg 00016 mmol) was dissolved in about 128 mL of THF and K2CO3 (aq)

(032 mL 02 M) was added The mixture was stirred for overnight Mixture was concentrated in vacuo

and analyzed by HPLCMS

A solution of iodine (7 mg 0027 mmol 5 eq) in CH3OH (4 mL ~10-3

M) was stirred vigorously

and compound 139 (50 mg 0005 mmol) compound 140 (8 mg 0004 mml) compound 141 (10 mg

0005 mmol) compound 74 (10 mg 0005 mmol) compound 78 (10 mg 0005 mmol) in about 1 mL of

CH3OH were respectively added The reactions were stirred for overnight and then were quenched by

the addition of aqueous ascorbate (02 M) in pH 40 citrate (02 M) buffer (4 mL) The colorless

mixtures extracted were poured into 11 saturated aqueous NaCl and CH2Cl2 The aqueous layer were

extracted with CH2Cl2 (3 x 10 mL) The organic phases recombined were concentrated in vacuo and the

crudes were purified by HPLCMS

108

FRONTESPIZIOpdf
- Dottorato di ricerca in Chimica
- - tesi dottorato_de cola chiara

1

INDEX

CHAPTER 1 INTRODUCTION 3 11 PEPTIDOMIMETICS 5 12 PEPTOIDS A PROMISING CLASS OF PEPTIDOMIMETICS 9 13 CONFORMATIONAL STUDIES OF PEPTOIDS 11 14 PEPTOIDSrsquo APPLICATIONS 14 15 PEPTOID SINTHESYS 39 16 SYNTHESYS OF PNA MONOMERS AND OLIGOMERS 41 17 AIMS OF THE WORK 49 CHAPTER 2 CARBOXYALKYL PEPTOID PNAS SYNTHESIS AND HYBRIDIZATION PROPERTIES 51 21 INTRODUCTION 51 22 RESULTS AND DISCUSSION 55 23 CONCLUSIONS 60 24 EXPERIMENTAL SECTION 60 CHAPTER 3 STRUCTURAL ANALYSIS OF CYCLOPEPTOIDS AND THEIR COMPLEXES 80 31 INTRODUCTION 80 32 RESULTS AND DISCUSSION 85 33 CONCLUSIONS 102 34 EXPERIMENTAL SECTION 103 CHAPTER 4 CATIONIC CYCLOPEPTOIDS AS POTENTIAL MACROCYCLIC NONVIRAL VECTORS 115 41 INTRODUCTION 115 42 RESULTS AND DISCUSSION 122 43 CONCLUSIONS 125 44 EXPERIMENTAL SECTION 125 CHAPTER 5 COMPLEXATION WITH GD(III) OF CARBOXYETHYL CYCLOPEPTOIDS AS POSSIBLE CONTRAST AGENTS

IN MRI 132 51 INTRODUCTION 132 52 LARIAT ETHER AND CLICK CHEMISTRY 135 53 RESULTS AND DISCUSSION 141 54 EXPERIMENTAL SECTION 145 CHAPTER 6 CYCLOPEPTOIDS AS MIMETIC OF NATURAL DEFENSINS 157 61 INTRODUCTION 157 62 RESULTS AND DISCUSSION 162 63 CONCLUSIONS 167 65 EXPERIMENTAL SECTION 167

2

DCM Dichloromethane

t-Bu terz-Butyl

THF Tetrahydrofuran

3

Chapter 1

1 Introduction

or in diagnostics

4

11 Peptidomimetics

affinities

5

literature)6

6

is possible

7

For some

folds11

16798ndash16807

8

interactions

A B

13525ndash13530

9

helical

(SMH) presumes

10

circular peptoids20

activity22

De Riccardis26

14)

11

neoformans27

12

+ and K

+)

21a and its ability

to promote Na+H

29 However

= 1ndash5 mM)

13

identified the

and pool synthesis

14

of 2733

protein degradation

15

and temperature13

50-51 In

helix bundle

29

able to form a Zn2+

complex

16

(30-34 figure 18)21a

+ and K

17

18

Kirshenbaum

centre

19

116)

DNA recognition

Drug discovery

20

1 RNA targeting

2 DNA targeting

3 Protein targeting

4 Cellular delivery

5 Pharmacology

Nanotechnology

Pre-RNA world

21

and

ways

22

The shift

DNA hybridization

23

showed a Tm

= 227 degC

grey bonds)

Chem 2009 6113

24

workers55

Figure 125)

NH

NN

NNH

N

O O O

BBB

X n

49

O O O

11)

groups

25

Cl HON

R

O Fmoc

ON

R

O

HN

R

O

HATU or PyBOP

Cl

HOBr

O

OBr

OR-NH2

O

HN

R

O

DIC

26

NH

NOH

OO

NH2

NH

NNH

OO

Deprotection

H2NN

NH

OO

NH

NOH

OO

CouplingNH

NNH

OO

NH

N

OO

First cleavage

NH2

HNH

N

OO

B

nPNA

B-PGs B-PGs

B-PGsB-PGs

PGt PGt

PGt

27

17 Aims of the work

NH

NN

N

O

Base

OOO

NH

N

BaseO

O

N

NH

O

HO50

NH

NN

N

O

Base

OOO

NH

N

BaseO

O

N

NH

O

HO 51

FmocN

NOH

N

NH

O

t-BuO

O

n 50 n = 151 n = 5

28

and 58)

N

OO

O

N

O

N

O

56

N

NN

OO

O

N

O

57

N

O

N

O ON

N

O

O58

cyclohexapeptoid 58

HON

H

O

HON

H

O

n=661n=659n=460

n n

58

29

N

NN

N

NNO

O

OO

O

H3N

2X CF3COO-

N

NN

N

O

H3N

NH3

H3N

4X CF3COO-

62 63

N

NN

N

NNO

O

OO

O

H3N

NH3

H3N

NH3

6X CF3COO-

64

cyclohexapeptoid 64

Gd3+

were evaluated

30

NN

OOO

OH

O

HO O

HO

O

OHO

N NN

O OO OMe

NNNO

OO

MeO

NN

OOO

OH

O

OH

O

HO O

HO

O

HO

O

OHO

NN

OOO

O

OH

O

HO

O

OHO

6566

HO

NN

NNH

OO

O

STr STr

NHBoc NHBoc68

N

NN

N

NNO

O

OO

O

NHBoc

SH

BocHN

HS

69N

NN

N

NNO

O

OO

O

NHBoc

S

BocHN

S

70

59

31

N

NN

N

O O

O

OOO

O

SH

HS

N

NN

N

O O

O

OO

O

S

72 73

NN

OO

O

STr

71

N

O

NH

STr

HO

OHNN

N

NN

OO

O

OO

O

TrS

NOO

N

NN

NNH

OO

O

TrS

74N

N

N N

NN

N

NO

O

O O O

OOO

O

NO

SH

HS

N

N N

NN

N

NO

O

O O O

OOO

O

NO

S

75

76

OH

NN

N

OO

O

S

NO

O

N

NH

O

S

77

32

HO

NN

OO O

OO

OTrS

78

NOO

N

HN

O

O STr

N

N N

NN

N

NO

O

O O O

OOO

O

NO

HS

79

SH

N

N N

NN

N

NO

O

O O O

OOO

O

NO

S

80

S

HO

NN

OO O

OO

O

S

NOO

N

HN

O

OS

81

79 80 and 81

33

Chapter 2

21 Introduction

Unfortunately

and poor

cellular uptake62

N-methyl content

the oligomeric

revealed an

and

34

lipids70

(decoy)72

2001 268 6066ndash6075

FmocN

NOH

N

NH

O

t-BuO

O

n 32 n = 133 n = 5

35

221 Chemistry

intermediate 87

36

Scheme 22) The

yields

O

t-BuONH2 O

OH+

O

t-BuON

R

82 83 84 R = H

85 R = Fmoc

a

b

c

d

O

t-BuON

Fmoc

O

t-BuON

Fmoc

OHOH

OHN

O

e

O

t-BuON

Fmoc NO

OR

86 87

O

NH

O

88 R = CH3

50 R = H

f

37

O

HO

NHCbz

89

5

O

t-BuO

NH2

5

a

INHCbz

9190

b

O

t-BuO

NHCbz

5

HONH2 HO

NHCbz

92 93 94

c d

e

O

t-BuO

N

5

NHR

95 R = H R = Cbz

R

h

97 R = Fmoc R = Hg

HN

O

t-BuO

N

5

Fmoc

51 R = H

98

i

NO

OR

O

t-BuO

N

5

Fmoc

l

ON

NH

O

91 94+

99 R = CH2CH3

38

duplexa

DNA mis-matchedb

(PNA sequence)8a

486 364

(DNA sequence)9

335 265

39

23 Conclusions

--negative

241 General Methods

400 (1H at 40013 MHz

1H at 25013 MHz

13C at 6289 MHz)

CDCl3 = 770 CD2HOD

= 334 13

40

242 Chemistry

664 716 827 1728 mz (ES) 206 (MH+) (HRES) MH

+ found 2061390 C9H20NO4

+ requires

2061392

831 1201 1202 1249 1252 1273 1279 1280 1415 1438 1564 1573 1704 1713 mz (ES)

428 (MH+) (HRES) MH

+ found 4282070 C24H30NO6

+ requires 4282073

41

C (7550 MHz CDCl3) 282 473 506 508 578 584 681 686 826 827 1202 1249 1252

1273 1280 1415 1438 1439 1560 1564 1686 1687 1987 mz (ES) 396 (MH+) (HRES) MH

+

505 533 672 676 815 817 1197 1247 1249 1268 1275 1410 1437 1560 1562 1687

1694 1719 1721 mz (ES) 469 (MH+) (HRES) MH

+ found 4692341 C26H33N2O6

+ requires

4692339

Compound 88

42

475 478 482 491 506 518 521 525 530 665 682 823 825 1105 1202 1245 1248

12514 1253 1273 1274 1275 1280 1281 1413 1414 1438 1440 1511 1564 1627 1644

1691 1692 mz (ES) 634 (MH+) (HRES) MH

+ found 6342767 C34H40N4O9

+ requires 6342765

Compound 50

CDCl3) 123 282 299 464 473 487 502 509 536 683 823 826 1108 1202 1249 1252

1253 1273 1275 1280 1414 1421 1438 1440 1517 1566 1650 1652 1682 1690 1692

1723 mz (ES) 620 (MH+) (HRES) MH

+ found 6202611 C33H38N3O9

+ requires 6202608

CDCl3) 245 260 280 295 353 408 664 799 1279 1280 1283 1366 1563 1728 mz (ES)

322 (MH+) (HRES) MH

+ found 3222015 C18H28NO4

+ requires 3222018

43

+ found 1881647 C10H22NO2

+ requires 1881651

1570 mz (ES) 196 (MH+) (HRES) MH

+ found 1960970 C10H14NO3

+ requires 1960974

+ found 3059989 C10H13INO2

+ requires 3059991

44

1279 1280 1283 1361 1566 1728 mz (ES) 365 (MH+) (HRES) MH

+ found 3652437

Compound (96)

392 396 462 468 471 476 663 797 1196 1244 1268 1274 1278 1282 1364 1411

1437 1556 1563 1727 mz (ES) 587 (MH+) (HRES) MH

+ found 5873120 C35H43N2O6

+ requires

5873121

Compound (97)

351 254 388 424 465 473 479 534 670 801 1198 1199 1240 1246 1269 1271 1277

1405 1413 1438 1490 1558 1571 1727 1729 mz (ES) 453 (MH+) (HRES) MH

+ found

4532740 C27H37N2O4+ requires 4532748

Compound (98)

45

1269 1275 1413 1440 1559 1562 1722 1729 mz (ES) 539 (MH+) (HRES) MH

+ found

5393117 C31H43N2O6+ requires 5393121

Compound (99)

246 260 280 353 464 467 472 478 481 507 617 669 801 1106 1110 1199 1246

1270 1276 1413 1438 1439 1512 1564 1643 1677 1689 1730 mz (ES) 705 (MH+)

+ requires 7053500

Compound (51)

46

458 478 485 488 496 547 683 814 816 1118 1119 1212 1259 1265 1283 1290

1295 1303 1391 1426 1429 1450 1451 1526 1577 1662 1690 1727 1744 mz (ES) 677

(MH+) (HRES) MH

+ found 6773185 C36H45N4O9

+ requires 6773187

800 1730 mz (ES) 262 (MH+) (HRES) MH

+ found 2622017 C13H28NO4

+ requires 2622018 101

293 294 367 568 569 583 591 660 662 705 710 815 1746 mz (ES) 336 (MH+) (HRES)

+ requires 3362386

O

t-BuO

NH2

5

91

O

OH

83

+

O

t-BuO

NR

OHOH

101 R =OH

OH

100 R = H

5

47

ndash 283911 60

ndash

48

Chapter 3

31 Introduction

as

the

102 103

49

hexamer (figure 33)

hydrogen bonding

50

crystal lattice

networks 81-82

51

58 (figure 36)

N

OO

O

N

O

N

O

56

N

NN

OO

O

N

O

57

N

O

N

O ON

N

O

O58

cyclohexapeptoid 58

321 Chemistry

(scheme 31)

52

Cl

HOBr

O

OBr

O

HON

H

O

HON

H

O

n=6 106

n=6 104n=4 105

NH2

ONH2

n

HON

NN

O

N

O

NNH

O

N

OO

O

N

O

N

O

PyBOP DIPEA DMF

104

56

80

HON

NN

O

NH

O

N

NN

OO

O

N

O

PyBOP DIPEA DMF

105

57

columns

53

HON

NN

O

N

O

NNH

O

O O O

O

106

N

O

N

O ON

N

O

O58

PyBOP DIPEA DMF

87

peptide sequences88

4 seeding

54

precipitate

crystals

8 CHCl3

with seeding

Needlelike

crystals

precipitate

vapor phase

Crystals

55

1 CH2Cl2 Slow

evaporation

Prismatic

crystals

2 CHCl3 Slow

evaporation

Precipitate

evaporation

Crystalline

Aggregates

4 CHCl3 Hexane Slow

evaporation

Little

crystals

1 CHCl3 Slow

evaporation

Crystals

2 CHCl3 CH3CN Slow

evaporation

Precipitate

3 AcOEt CH3CN Slow

evaporation

Precipitate

5 AcOEt CH3CN Slow

evaporation

Prismatic

crystals

6 CH3CN i-PrOH Slow

evaporation

Little

crystals

7 CH3CN MeOH Slow

evaporation

Crystalline

precipitate

between two

phases

Precipitate

9 CH3CN Crystallin

precipitate

56

56A 56B

57

58

57

PM (g mol-1

) 91903 88303 58869 51336

Source Rotating

anode

Rotating

anode

Rotating

anode

Rotating

anode

λ (Aring)

154178 154178 154178 154178

a (Aring)

b (Aring)

c (Aring)

α (deg)

β (deg)

γ (deg)

4573(7)

9283(14)

2383(4)

10597(4)

9240(12)

11581(13)

11877(17)

10906(2)

10162(5)

92170(8)

10899(3)

10055(3)

27255(7)

8805(3)

11014(2)

12477(2)

7097(2)

77347(16)

8975(2)

V (Aring3 ) 9725(27) 1169(3) 29869(14) 11131(5)

Z 8 1 4 2

Dcalc (g cm-3

) 1206 1254 1309 1532

58

μ (cm-1

) 0638 0663 0692 2105

Observed

reflecti

on (Igt2I )

4883 1856 1985 1841

R1 (Igt2I) 01345 00958 00586 01165

Rw 04010 03137 02208 03972

sides

56A

59

space group is P1

inversion centre

60

arsquo 0 1 0 a b

crsquo 1 0 0 c a

structure of 56B

61

62

method (figure 315)

(a)

63

(b)

64

34 Conclusions

were reported

351 General Methods

400 (1H at 40013 MHz

1H at 25013 MHz

13C at 6289 MHz)

CDCl3 = 770 CD2HOD

= 334 13

65

352 Synthesis

approach11

+ found 9014290 C54H57N6O7

+ requires

9014289 100

+ found 6072925 C36H39N4O5

+ requires

6062920 100

+ found 7093986 C30H57N6O13

+ requires

7093984 100

66

atmosphere

+ found

8834110 C54H55N6O6+

+ found 5892740 C36H37N4O4

180 min

rotamers) 171 5 1710 1796 1701 1700 1698 1697 1695 1693 1691 1689 1687 1682

1681 717 715 714 713 710 708 706 (bs) 702 (bs) 700 (bs) 698 (bs)695 (bs) 693 (bs)

67

+ found 6913810 C30H55N6O12

Data reduction

0)(

cFFw

2

0

22

0

2

iii

iciii

Fw

FFwwR

Goumlttingen 1997

68

Rietveld analysis

56B

Data reduction

92

0

1

ii

icii

F

FFR

69

Data reduction

(154178 Aring)

Data reduction

70

Chapter 4

41 Introduction

98mdash that

efficiency

polylysine3101

) to synthetic

)

71

dendrimers113

nanoparticles114

60 452ndash472

72

108

examined a set

73

chains

N

NN

N

NNO

O

OO

O

H3N

2X CF3COO-

N

NN

N

O

H3N

NH3

H3N

4X CF3COO-

62 63

74

N

NN

N

NNO

O

OO

O

H3N

NH3

H3N

NH3

6X CF3COO-

64

cyclohexapeptoid 64

421 Synthesis

NH2

CH3OH Et3N

NH2

NH

O

110

111

O O O

O O

(Boc)2O

Cl

HOBr

O

OBr

O

NH2

NH2BocHN

111

75

HON

O

N

ONHBoc6

N

H

ONHBoc

6

2

N

H

ONHBoc

6

6HO

113

114

HON

O

N

O

N

H

ONHBoc

6

2112

HON

O

N

O

NH

ONHBoc

6

2

112

HATU DIPEA

DMF 33N

NN

N

NN

O O

O

OO

O

NHO

O

HN

O

115

76

HON

O

N

ONHBoc6

NH

ONHBoc6

2

113

N

NN

N

O

HN

NH

O

OO

O

HN

O O

116

HATU DIPEA

DMF 33

NH

ONHBoc6

6HO114

N

NN

N

NNO

O

OO

O

HNNH

HN

OO

NHO

O

NH

O

NH

O

O O

O

117

HATU DIPEA

DMF 24

77

43 Conclusions

441 Synthesis

Compound 111

+ found 2171920 C11H25N2O2

+ requires

2171916

approach11

78

+ found 11196485

C62H87N8O11+ requires 11196489 100

+ found 13378690

C70H117N10O15+ requires 13378694 100

+ found 15560910

C78H147N12O19+ requires 15560900 100

79

33 mz (ES) 1101 (MH+) (HRES) MH

+ found 11013785 C62H85N8O10

tR 206 min

conformers) 1706 1690 1689 15863 1561 1558 1557 1444 1434 1433 1408 1407 1362

1360 1279 1275 1274 1269 1264 1245 1194 817 816 675 670 660 659 598 504

500 499 487 483 467 466 390 274 205 33 mz (ES) 1319 (MH+) (HRES) MH

+ found

+ found 15380480 C78H145N12O18

225 min

1371 (bs) 1311 (bs) 1302 1297 1293 1290 1286 1283 1270 548 538 528 521 (bs) 508

3+ requires 9161797

80

+) (HRES) MH

+ found 9232792

C50H87N10O65+

requires 9232792

+ found 9433978 C48H103N12O6

7+ requires 9433970

81

Chapter 5

51 Introduction

The image

82

uncontrasted images

s-1

(20 MHz and

39degC)2

where R1

is the

1p + Ros

127

N N

NNHOOC

HOOC

COOH

(DOTA)

119

NH

N NH

N

COOH

CONHCH3H3CHNOC

HOOC

DTPA-BMA

121

N N

NNHOOC

HOOC

COOH

OH

CH3HP-DO3A

120

DTPA

NH

N NH

N

COOH

COOHHOOC

HOOC

118

83

T1M (eq 52)

52 Eq )τ(555

][

51 Eq

1

1111

MM

is

p

os

p

is

p

oobs

T

CqR

RRRR

53 Eq τω1

7τ

τω1

3τ1)S(S

r

γγ

4π

μ

15

2

T

12

c2

2

s

c2

2

c1

2

H

c1

6

GdH

2

H

2

s

22

0

1M

54 Eq τ

1

τ

1

τ

1

τ

1

EMRci i

molecules

1p and Ros

design (figure 52)1

84

NN

OOO

OH

O

HO O

HO

O

OHO

N NN

O OO OMe

NNNO

OO

MeO

NN

OOO

OH

O

OH

O

HO O

HO

O

HO

O

OHO

NN

OOO

O

OH

O

HO

O

OHO

6566

85

Na+ Ca

2+ and NH

128

of

The

synthesis131

N N NR

H

R

N

N N

R

H N

N N

R

H

R

128

Chem 2002 67 3057 131

Int Ed 2002 41 1053 132

86

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

122

531 Synthesis

O

Br -Cl+H3N O

O

OHN O

O

DIPEA DMF

18 h rt

O

Cl

O Fmoc-Cl =

HO

O

N O

O

Fmoc

123 124125

DIPEA = N

126

127 128 and 129 (figure 55)

HON

O

O Ot-Bu

H

6127

HON

O

O Ot-Bu

3N

O

H

OMe128

HON

O

O Ot-Bu

2

N

O

N

O

H

Ot-BuO

129

87

HATU DIPEA

DMF 654

NN

N

NN

OO

O

Ot-Bu

O

Ot-Bu

O

t-BuO O

t-BuO

O

t-BuO

O

Ot-BuO

HON

O

O Ot-Bu

H

6

127

130

HON

O

O Ot-Bu

3

N

O

H

OMe128

NN

N

NN

OO

O

Ot-Bu

O

t-BuO

O

Ot-BuO

HATU DIPEA

DMF 82

131

HON

O

O Ot-Bu

2

N

O

N

O

H

Ot-BuO

129

HATU DIPEA

DMF 71

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

122

88

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

CuSO4 5H2O

sodium ascorbate

H2OCH3OH

N NN

O O

O OMe

NNNO

OO

MeO

NO

OO

NN OMe2

53

122

133

132

afford 67

excretion

figure 57)

89

CP = cyclopeptoid

which was 315 mM-1

s-1

e 253 mM-1

s-1

)

the

134

90

2 (s

Gd-65 21times1019

275 1times10-8

280 3 15

Gd-66 28times1019

225 1times10-8

216 3 14

coordination sphere

541 Synthesis

Compound 125

91

1401 2797 3350 4430 4914 6041 8144 16888 17077 mz (ES) 232 (MH+) (HRES) MH+

Compound 126

17111 17161 17488 17504 mz (ES) 454 (MH+) (HRES) MH

+ found 454229 C26H32NO6

+

requires 454223

92

+ found 1129 6500

C54H93N6O19+ requires 11296425 80

+ found 9195248

C42H75N6O16+ requires 9195240 75

+ found 9495138

C43H73N6O15+ requires 9495134 85

93

4531 4555 4572 4594 4676 4756 4774 4858 4896 4977 5004 5037 5102 5277 5323

5340 8058 8141 16765 16776 16793 16819 16832 16856 16886 16890 16902 16915

16928 16938 16951 16993 17027 17038 17062 17069 17078 17091 17096 17114

17134 17139 17158 17167 17174 17187 65 mz (ES) 1111 (MH+) (HRES) MH

+ found

11116395 C54H91N6O18+

solution 40 mM) 2846 3478 4550 5020 8227 12724 12822 14310 17015 17173

4962 4992 5061 5397 5993 6026 7280 8195 8110 8269 11401 11800 16061 16072

17001 17075 17121 17145 17190 17219 17245 17288 17310 82 mz (ES) 901 (MH+)

3653 3671 3698 4378 4402 4421 4437 4509 4687 4755 4767 4780 4866 4903 4925

4991 5016 5053 5091 5297 5329 7236 7250 7286 8056 8127 8136 15786 15863

16720 16805 16851 16889 17026 17100 17151 17152 71 mz (ES) 931 (MH+) (HRES)

MH+ found 9315029 C46H71N6O14

94

4301 4448 4501 4564 4838 4891 4944 5060 5334 5892 6915 6999 7052 7189 8054

8069 8080 8092 8136 11387 11640 12416 12435 12446 12465 12474 12532 12547

14221 15891 15941 15952 16872 16954 17140 17055 17100 17120 17131 mz (ES) 1397

(MH+) (HRES) MH

+ found 13977780 C64H109N12O22

4531 4555 4572 4594 4676 4756 4774 4858 4896 4977 5004 5037 5102 5277 5323

5340 8058 8141 16765 16776 16793 16819 16832 16856 16886 16890 16902 16915

16928 16938 16951 16993 17027 17038 17062 17069 17078 17091 17096 17114

17134 17139 17158 17167 17174 17187 mz (ES) 775 (MH+) (HRES) MH

+ found 7752638

C30H43N6O18+

rotamers) 1459 3143 3214 3258 4359 4400 4460 4504 4574 4931 4969 5004 5757

5777 5785 5802 5829 6551 6942 6957 7011 7021 7035 16870 16891 16898 16934

16945 16957 16987 16997 17035 17083 17110 17160 17182 17275 17298 17318

95

17330 mz (ES) 733 (MH+) (HRES) MH

+ found 7333259 C30H49N6O15

tR 843 min

96

Chapter 6

61 Introduction

cell death137

α-defensin

97

β-defensin

θ-defensin

The anti-HIV-1

or

98

HO

NN

NNH

OO

O

STr STr

NHBoc NHBoc68

N

NN

N

NNO

O

OO

O

NHBoc

SH

BocHN

HS

69N

NN

N

NNO

O

OO

O

NHBoc

S

BocHN

S

70

N

NN

N

O O

O

OOO

O

SH

HS

N

NN

N

O O

O

OO

O

S

72 73

NN

OO

O

STr

71

N

O

NH

STr

HO

99

OHNN

N

NN

OO

O

OO

O

TrS

NOO

N

NN

NNH

OO

O

TrS

74N

N

N N

NN

N

NO

O

O O O

OOO

O

NO

SH

HS

N

N N

NN

N

NO

O

O O O

OOO

O

NO

S

75

76

OH

NN

N

OO

O

S

NO

O

N

NH

O

S

77

HO

NN

OO O

OO

OTrS

78

NOO

N

HN

O

O STr

N

N N

NN

N

NO

O

O O O

OOO

O

NO

HS

79

SH

N

N N

NN

N

NO

O

O O O

OOO

O

NO

S

80

S

HO

NN

OO O

OO

O

S

NOO

N

HN

O

OS

81

80 and 81

100

conditions10

bond formation145

10

621 Synthesis

and the

NH2

CH3OH Et3N

H2NNH

O

134 135O O O

O O

(Boc)2O

NH2

SHH2N

S(Ph)3COH

TFA rt quant

136 137

101

HPLCMS analysis149

N

NN

N

O O

O

OO

O

STr

TrS

139

N

NN

N

NNO

O

NHBoc

STr

BocHN

TrS

138

N

N N

NN

N

O

O O O

OO

O

N

O

NO

STr

TrS

140

N

N N

N

O

O O O

OO

O

N

O

NO

TrS

141 STr

acid151

102

in a protic solvent

61

118 7237 154

138

1

2

3

4

CuCl (40) H2O20

TFA H2O Et3SiH

(925525)17

I2 (5 eq)16

DMSO (5) DIPEA19

CH2Cl2

TFA

AcOHH2O (41)

CH3CN

-

139

5

6

7

8

9

TFA H2O Et3SiH

(925525)17

DMSO (5) DIPEA19

DMSO (5) DBU19

K2CO3 (02 M)

I2 (5 eq)154

TFA

CH3CN

THF

CH3OH

-

140

9 I2 (5 eq)154

CH3OH gt70

141

9 I2 (5 eq)154

CH3OH gt70

74

9 I2 (5 eq)154

CH3OH gt70

78

9 I2 (5 eq)154

CH3OH gt70

103

closer

63 Conclusions

641 Synthesis

Compound 135

(MH+) (HRES) MH

+ found 1891600 C9H21N2O2

+ requires 1891598

104

Compound 137

+ found 3201470

approach11

+ found 14197497

C80H107N8O11S2+ requires 14197495 100

+ found 14157912

C82H111N8O9S2+ requires 14157910 100

105

+ found 18681278

C106H155N12O13S2+ requires 18681273 100

+ found 18360650

C104H147N12O13S2+ requires 18360647 100

+ found 14017395 C80H105N8O10S2

+ requires

14017390 HPLC tR 250 min

106

+ found 13977810 C82H109N8O8S2

+ requires

13977804 HPLC tR 271 min

+ found 18501170 C106H153N12O12S2

+ requires

18501167 HPLC tR 330 min

+ found 18040390

75-76-77 and 79-80-81)

HPLCMS

107

108

FRONTESPIZIOpdf

2

DCM Dichloromethane

t-Bu terz-Butyl

THF Tetrahydrofuran

3

Chapter 1

1 Introduction

or in diagnostics

4

11 Peptidomimetics

affinities

5

literature)6

6

is possible

7

For some

folds11

16798ndash16807

8

interactions

A B

13525ndash13530

9

helical

(SMH) presumes

10

circular peptoids20

activity22

De Riccardis26

14)

11

neoformans27

12

+ and K

+)

21a and its ability

to promote Na+H

29 However

= 1ndash5 mM)

13

identified the

and pool synthesis

14

of 2733

protein degradation

15

and temperature13

50-51 In

helix bundle

29

able to form a Zn2+

complex

16

(30-34 figure 18)21a

+ and K

17

18

Kirshenbaum

centre

19

116)

DNA recognition

Drug discovery

20

1 RNA targeting

2 DNA targeting

3 Protein targeting

4 Cellular delivery

5 Pharmacology

Nanotechnology

Pre-RNA world

21

and

ways

22

The shift

DNA hybridization

23

showed a Tm

= 227 degC

grey bonds)

Chem 2009 6113

24

workers55

Figure 125)

NH

NN

NNH

N

O O O

BBB

X n

49

O O O

11)

groups

25

Cl HON

R

O Fmoc

ON

R

O

HN

R

O

HATU or PyBOP

Cl

HOBr

O

OBr

OR-NH2

O

HN

R

O

DIC

26

NH

NOH

OO

NH2

NH

NNH

OO

Deprotection

H2NN

NH

OO

NH

NOH

OO

CouplingNH

NNH

OO

NH

N

OO

First cleavage

NH2

HNH

N

OO

B

nPNA

B-PGs B-PGs

B-PGsB-PGs

PGt PGt

PGt

27

17 Aims of the work

NH

NN

N

O

Base

OOO

NH

N

BaseO

O

N

NH

O

HO50

NH

NN

N

O

Base

OOO

NH

N

BaseO

O

N

NH

O

HO 51

FmocN

NOH

N

NH

O

t-BuO

O

n 50 n = 151 n = 5

28

and 58)

N

OO

O

N

O

N

O

56

N

NN

OO

O

N

O

57

N

O

N

O ON

N

O

O58

cyclohexapeptoid 58

HON

H

O

HON

H

O

n=661n=659n=460

n n

58

29

N

NN

N

NNO

O

OO

O

H3N

2X CF3COO-

N

NN

N

O

H3N

NH3

H3N

4X CF3COO-

62 63

N

NN

N

NNO

O

OO

O

H3N

NH3

H3N

NH3

6X CF3COO-

64

cyclohexapeptoid 64

Gd3+

were evaluated

30

NN

OOO

OH

O

HO O

HO

O

OHO

N NN

O OO OMe

NNNO

OO

MeO

NN

OOO

OH

O

OH

O

HO O

HO

O

HO

O

OHO

NN

OOO

O

OH

O

HO

O

OHO

6566

HO

NN

NNH

OO

O

STr STr

NHBoc NHBoc68

N

NN

N

NNO

O

OO

O

NHBoc

SH

BocHN

HS

69N

NN

N

NNO

O

OO

O

NHBoc

S

BocHN

S

70

59

31

N

NN

N

O O

O

OOO

O

SH

HS

N

NN

N

O O

O

OO

O

S

72 73

NN

OO

O

STr

71

N

O

NH

STr

HO

OHNN

N

NN

OO

O

OO

O

TrS

NOO

N

NN

NNH

OO

O

TrS

74N

N

N N

NN

N

NO

O

O O O

OOO

O

NO

SH

HS

N

N N

NN

N

NO

O

O O O

OOO

O

NO

S

75

76

OH

NN

N

OO

O

S

NO

O

N

NH

O

S

77

32

HO

NN

OO O

OO

OTrS

78

NOO

N

HN

O

O STr

N

N N

NN

N

NO

O

O O O

OOO

O

NO

HS

79

SH

N

N N

NN

N

NO

O

O O O

OOO

O

NO

S

80

S

HO

NN

OO O

OO

O

S

NOO

N

HN

O

OS

81

79 80 and 81

33

Chapter 2

21 Introduction

Unfortunately

and poor

cellular uptake62

N-methyl content

the oligomeric

revealed an

and

34

lipids70

(decoy)72

2001 268 6066ndash6075

FmocN

NOH

N

NH

O

t-BuO

O

n 32 n = 133 n = 5

35

221 Chemistry

intermediate 87

36

Scheme 22) The

yields

O

t-BuONH2 O

OH+

O

t-BuON

R

82 83 84 R = H

85 R = Fmoc

a

b

c

d

O

t-BuON

Fmoc

O

t-BuON

Fmoc

OHOH

OHN

O

e

O

t-BuON

Fmoc NO

OR

86 87

O

NH

O

88 R = CH3

50 R = H

f

37

O

HO

NHCbz

89

5

O

t-BuO

NH2

5

a

INHCbz

9190

b

O

t-BuO

NHCbz

5

HONH2 HO

NHCbz

92 93 94

c d

e

O

t-BuO

N

5

NHR

95 R = H R = Cbz

R

h

97 R = Fmoc R = Hg

HN

O

t-BuO

N

5

Fmoc

51 R = H

98

i

NO

OR

O

t-BuO

N

5

Fmoc

l

ON

NH

O

91 94+

99 R = CH2CH3

38

duplexa

DNA mis-matchedb

(PNA sequence)8a

486 364

(DNA sequence)9

335 265

39

23 Conclusions

--negative

241 General Methods

400 (1H at 40013 MHz

1H at 25013 MHz

13C at 6289 MHz)

CDCl3 = 770 CD2HOD

= 334 13

40

242 Chemistry

664 716 827 1728 mz (ES) 206 (MH+) (HRES) MH

+ found 2061390 C9H20NO4

+ requires

2061392

831 1201 1202 1249 1252 1273 1279 1280 1415 1438 1564 1573 1704 1713 mz (ES)

428 (MH+) (HRES) MH

+ found 4282070 C24H30NO6

+ requires 4282073

41

C (7550 MHz CDCl3) 282 473 506 508 578 584 681 686 826 827 1202 1249 1252

1273 1280 1415 1438 1439 1560 1564 1686 1687 1987 mz (ES) 396 (MH+) (HRES) MH

+

505 533 672 676 815 817 1197 1247 1249 1268 1275 1410 1437 1560 1562 1687

1694 1719 1721 mz (ES) 469 (MH+) (HRES) MH

+ found 4692341 C26H33N2O6

+ requires

4692339

Compound 88

42

475 478 482 491 506 518 521 525 530 665 682 823 825 1105 1202 1245 1248

12514 1253 1273 1274 1275 1280 1281 1413 1414 1438 1440 1511 1564 1627 1644

1691 1692 mz (ES) 634 (MH+) (HRES) MH

+ found 6342767 C34H40N4O9

+ requires 6342765

Compound 50

CDCl3) 123 282 299 464 473 487 502 509 536 683 823 826 1108 1202 1249 1252

1253 1273 1275 1280 1414 1421 1438 1440 1517 1566 1650 1652 1682 1690 1692

1723 mz (ES) 620 (MH+) (HRES) MH

+ found 6202611 C33H38N3O9

+ requires 6202608

CDCl3) 245 260 280 295 353 408 664 799 1279 1280 1283 1366 1563 1728 mz (ES)

322 (MH+) (HRES) MH

+ found 3222015 C18H28NO4

+ requires 3222018

43

+ found 1881647 C10H22NO2

+ requires 1881651

1570 mz (ES) 196 (MH+) (HRES) MH

+ found 1960970 C10H14NO3

+ requires 1960974

+ found 3059989 C10H13INO2

+ requires 3059991

44

1279 1280 1283 1361 1566 1728 mz (ES) 365 (MH+) (HRES) MH

+ found 3652437

Compound (96)

392 396 462 468 471 476 663 797 1196 1244 1268 1274 1278 1282 1364 1411

1437 1556 1563 1727 mz (ES) 587 (MH+) (HRES) MH

+ found 5873120 C35H43N2O6

+ requires

5873121

Compound (97)

351 254 388 424 465 473 479 534 670 801 1198 1199 1240 1246 1269 1271 1277

1405 1413 1438 1490 1558 1571 1727 1729 mz (ES) 453 (MH+) (HRES) MH

+ found

4532740 C27H37N2O4+ requires 4532748

Compound (98)

45

1269 1275 1413 1440 1559 1562 1722 1729 mz (ES) 539 (MH+) (HRES) MH

+ found

5393117 C31H43N2O6+ requires 5393121

Compound (99)

246 260 280 353 464 467 472 478 481 507 617 669 801 1106 1110 1199 1246

1270 1276 1413 1438 1439 1512 1564 1643 1677 1689 1730 mz (ES) 705 (MH+)

+ requires 7053500

Compound (51)

46

458 478 485 488 496 547 683 814 816 1118 1119 1212 1259 1265 1283 1290

1295 1303 1391 1426 1429 1450 1451 1526 1577 1662 1690 1727 1744 mz (ES) 677

(MH+) (HRES) MH

+ found 6773185 C36H45N4O9

+ requires 6773187

800 1730 mz (ES) 262 (MH+) (HRES) MH

+ found 2622017 C13H28NO4

+ requires 2622018 101

293 294 367 568 569 583 591 660 662 705 710 815 1746 mz (ES) 336 (MH+) (HRES)

+ requires 3362386

O

t-BuO

NH2

5

91

O

OH

83

+

O

t-BuO

NR

OHOH

101 R =OH

OH

100 R = H

5

47

ndash 283911 60

ndash

48

Chapter 3

31 Introduction

as

the

102 103

49

hexamer (figure 33)

hydrogen bonding

50

crystal lattice

networks 81-82

51

58 (figure 36)

N

OO

O

N

O

N

O

56

N

NN

OO

O

N

O

57

N

O

N

O ON

N

O

O58

cyclohexapeptoid 58

321 Chemistry

(scheme 31)

52

Cl

HOBr

O

OBr

O

HON

H

O

HON

H

O

n=6 106

n=6 104n=4 105

NH2

ONH2

n

HON

NN

O

N

O

NNH

O

N

OO

O

N

O

N

O

PyBOP DIPEA DMF

104

56

80

HON

NN

O

NH

O

N

NN

OO

O

N

O

PyBOP DIPEA DMF

105

57

columns

53

HON

NN

O

N

O

NNH

O

O O O

O

106

N

O

N

O ON

N

O

O58

PyBOP DIPEA DMF

87

peptide sequences88

4 seeding

54

precipitate

crystals

8 CHCl3

with seeding

Needlelike

crystals

precipitate

vapor phase

Crystals

55

1 CH2Cl2 Slow

evaporation

Prismatic

crystals

2 CHCl3 Slow

evaporation

Precipitate

evaporation

Crystalline

Aggregates

4 CHCl3 Hexane Slow

evaporation

Little

crystals

1 CHCl3 Slow

evaporation

Crystals

2 CHCl3 CH3CN Slow

evaporation

Precipitate

3 AcOEt CH3CN Slow

evaporation

Precipitate

5 AcOEt CH3CN Slow

evaporation

Prismatic

crystals

6 CH3CN i-PrOH Slow

evaporation

Little

crystals

7 CH3CN MeOH Slow

evaporation

Crystalline

precipitate

between two

phases

Precipitate

9 CH3CN Crystallin

precipitate

56

56A 56B

57

58

57

PM (g mol-1

) 91903 88303 58869 51336

Source Rotating

anode

Rotating

anode

Rotating

anode

Rotating

anode

λ (Aring)

154178 154178 154178 154178

a (Aring)

b (Aring)

c (Aring)

α (deg)

β (deg)

γ (deg)

4573(7)

9283(14)

2383(4)

10597(4)

9240(12)

11581(13)

11877(17)

10906(2)

10162(5)

92170(8)

10899(3)

10055(3)

27255(7)

8805(3)

11014(2)

12477(2)

7097(2)

77347(16)

8975(2)

V (Aring3 ) 9725(27) 1169(3) 29869(14) 11131(5)

Z 8 1 4 2

Dcalc (g cm-3

) 1206 1254 1309 1532

58

μ (cm-1

) 0638 0663 0692 2105

Observed

reflecti

on (Igt2I )

4883 1856 1985 1841

R1 (Igt2I) 01345 00958 00586 01165

Rw 04010 03137 02208 03972

sides

56A

59

space group is P1

inversion centre

60

arsquo 0 1 0 a b

crsquo 1 0 0 c a

structure of 56B

61

62

method (figure 315)

(a)

63

(b)

64

34 Conclusions

were reported

351 General Methods

400 (1H at 40013 MHz

1H at 25013 MHz

13C at 6289 MHz)

CDCl3 = 770 CD2HOD

= 334 13

65

352 Synthesis

approach11

+ found 9014290 C54H57N6O7

+ requires

9014289 100

+ found 6072925 C36H39N4O5

+ requires

6062920 100

+ found 7093986 C30H57N6O13

+ requires

7093984 100

66

atmosphere

+ found

8834110 C54H55N6O6+

+ found 5892740 C36H37N4O4

180 min

rotamers) 171 5 1710 1796 1701 1700 1698 1697 1695 1693 1691 1689 1687 1682

1681 717 715 714 713 710 708 706 (bs) 702 (bs) 700 (bs) 698 (bs)695 (bs) 693 (bs)

67

+ found 6913810 C30H55N6O12

Data reduction

0)(

cFFw

2

0

22

0

2

iii

iciii

Fw

FFwwR

Goumlttingen 1997

68

Rietveld analysis

56B

Data reduction

92

0

1

ii

icii

F

FFR

69

Data reduction

(154178 Aring)

Data reduction

70

Chapter 4

41 Introduction

98mdash that

efficiency

polylysine3101

) to synthetic

)

71

dendrimers113

nanoparticles114

60 452ndash472

72

108

examined a set

73

chains

N

NN

N

NNO

O

OO

O

H3N

2X CF3COO-

N

NN

N

O

H3N

NH3

H3N

4X CF3COO-

62 63

74

N

NN

N

NNO

O

OO

O

H3N

NH3

H3N

NH3

6X CF3COO-

64

cyclohexapeptoid 64

421 Synthesis

NH2

CH3OH Et3N

NH2

NH

O

110

111

O O O

O O

(Boc)2O

Cl

HOBr

O

OBr

O

NH2

NH2BocHN

111

75

HON

O

N

ONHBoc6

N

H

ONHBoc

6

2

N

H

ONHBoc

6

6HO

113

114

HON

O

N

O

N

H

ONHBoc

6

2112

HON

O

N

O

NH

ONHBoc

6

2

112

HATU DIPEA

DMF 33N

NN

N

NN

O O

O

OO

O

NHO

O

HN

O

115

76

HON

O

N

ONHBoc6

NH

ONHBoc6

2

113

N

NN

N

O

HN

NH

O

OO

O

HN

O O

116

HATU DIPEA

DMF 33

NH

ONHBoc6

6HO114

N

NN

N

NNO

O

OO

O

HNNH

HN

OO

NHO

O

NH

O

NH

O

O O

O

117

HATU DIPEA

DMF 24

77

43 Conclusions

441 Synthesis

Compound 111

+ found 2171920 C11H25N2O2

+ requires

2171916

approach11

78

+ found 11196485

C62H87N8O11+ requires 11196489 100

+ found 13378690

C70H117N10O15+ requires 13378694 100

+ found 15560910

C78H147N12O19+ requires 15560900 100

79

33 mz (ES) 1101 (MH+) (HRES) MH

+ found 11013785 C62H85N8O10

tR 206 min

conformers) 1706 1690 1689 15863 1561 1558 1557 1444 1434 1433 1408 1407 1362

1360 1279 1275 1274 1269 1264 1245 1194 817 816 675 670 660 659 598 504

500 499 487 483 467 466 390 274 205 33 mz (ES) 1319 (MH+) (HRES) MH

+ found

+ found 15380480 C78H145N12O18

225 min

1371 (bs) 1311 (bs) 1302 1297 1293 1290 1286 1283 1270 548 538 528 521 (bs) 508

3+ requires 9161797

80

+) (HRES) MH

+ found 9232792

C50H87N10O65+

requires 9232792

+ found 9433978 C48H103N12O6

7+ requires 9433970

81

Chapter 5

51 Introduction

The image

82

uncontrasted images

s-1

(20 MHz and

39degC)2

where R1

is the

1p + Ros

127

N N

NNHOOC

HOOC

COOH

(DOTA)

119

NH

N NH

N

COOH

CONHCH3H3CHNOC

HOOC

DTPA-BMA

121

N N

NNHOOC

HOOC

COOH

OH

CH3HP-DO3A

120

DTPA

NH

N NH

N

COOH

COOHHOOC

HOOC

118

83

T1M (eq 52)

52 Eq )τ(555

][

51 Eq

1

1111

MM

is

p

os

p

is

p

oobs

T

CqR

RRRR

53 Eq τω1

7τ

τω1

3τ1)S(S

r

γγ

4π

μ

15

2

T

12

c2

2

s

c2

2

c1

2

H

c1

6

GdH

2

H

2

s

22

0

1M

54 Eq τ

1

τ

1

τ

1

τ

1

EMRci i

molecules

1p and Ros

design (figure 52)1

84

NN

OOO

OH

O

HO O

HO

O

OHO

N NN

O OO OMe

NNNO

OO

MeO

NN

OOO

OH

O

OH

O

HO O

HO

O

HO

O

OHO

NN

OOO

O

OH

O

HO

O

OHO

6566

85

Na+ Ca

2+ and NH

128

of

The

synthesis131

N N NR

H

R

N

N N

R

H N

N N

R

H

R

128

Chem 2002 67 3057 131

Int Ed 2002 41 1053 132

86

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

122

531 Synthesis

O

Br -Cl+H3N O

O

OHN O

O

DIPEA DMF

18 h rt

O

Cl

O Fmoc-Cl =

HO

O

N O

O

Fmoc

123 124125

DIPEA = N

126

127 128 and 129 (figure 55)

HON

O

O Ot-Bu

H

6127

HON

O

O Ot-Bu

3N

O

H

OMe128

HON

O

O Ot-Bu

2

N

O

N

O

H

Ot-BuO

129

87

HATU DIPEA

DMF 654

NN

N

NN

OO

O

Ot-Bu

O

Ot-Bu

O

t-BuO O

t-BuO

O

t-BuO

O

Ot-BuO

HON

O

O Ot-Bu

H

6

127

130

HON

O

O Ot-Bu

3

N

O

H

OMe128

NN

N

NN

OO

O

Ot-Bu

O

t-BuO

O

Ot-BuO

HATU DIPEA

DMF 82

131

HON

O

O Ot-Bu

2

N

O

N

O

H

Ot-BuO

129

HATU DIPEA

DMF 71

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

122

88

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

CuSO4 5H2O

sodium ascorbate

H2OCH3OH

N NN

O O

O OMe

NNNO

OO

MeO

NO

OO

NN OMe2

53

122

133

132

afford 67

excretion

figure 57)

89

CP = cyclopeptoid

which was 315 mM-1

s-1

e 253 mM-1

s-1

)

the

134

90

2 (s

Gd-65 21times1019

275 1times10-8

280 3 15

Gd-66 28times1019

225 1times10-8

216 3 14

coordination sphere

541 Synthesis

Compound 125

91

1401 2797 3350 4430 4914 6041 8144 16888 17077 mz (ES) 232 (MH+) (HRES) MH+

Compound 126

17111 17161 17488 17504 mz (ES) 454 (MH+) (HRES) MH

+ found 454229 C26H32NO6

+

requires 454223

92

+ found 1129 6500

C54H93N6O19+ requires 11296425 80

+ found 9195248

C42H75N6O16+ requires 9195240 75

+ found 9495138

C43H73N6O15+ requires 9495134 85

93

4531 4555 4572 4594 4676 4756 4774 4858 4896 4977 5004 5037 5102 5277 5323

5340 8058 8141 16765 16776 16793 16819 16832 16856 16886 16890 16902 16915

16928 16938 16951 16993 17027 17038 17062 17069 17078 17091 17096 17114

17134 17139 17158 17167 17174 17187 65 mz (ES) 1111 (MH+) (HRES) MH

+ found

11116395 C54H91N6O18+

solution 40 mM) 2846 3478 4550 5020 8227 12724 12822 14310 17015 17173

4962 4992 5061 5397 5993 6026 7280 8195 8110 8269 11401 11800 16061 16072

17001 17075 17121 17145 17190 17219 17245 17288 17310 82 mz (ES) 901 (MH+)

3653 3671 3698 4378 4402 4421 4437 4509 4687 4755 4767 4780 4866 4903 4925

4991 5016 5053 5091 5297 5329 7236 7250 7286 8056 8127 8136 15786 15863

16720 16805 16851 16889 17026 17100 17151 17152 71 mz (ES) 931 (MH+) (HRES)

MH+ found 9315029 C46H71N6O14

94

4301 4448 4501 4564 4838 4891 4944 5060 5334 5892 6915 6999 7052 7189 8054

8069 8080 8092 8136 11387 11640 12416 12435 12446 12465 12474 12532 12547

14221 15891 15941 15952 16872 16954 17140 17055 17100 17120 17131 mz (ES) 1397

(MH+) (HRES) MH

+ found 13977780 C64H109N12O22

4531 4555 4572 4594 4676 4756 4774 4858 4896 4977 5004 5037 5102 5277 5323

5340 8058 8141 16765 16776 16793 16819 16832 16856 16886 16890 16902 16915

16928 16938 16951 16993 17027 17038 17062 17069 17078 17091 17096 17114

17134 17139 17158 17167 17174 17187 mz (ES) 775 (MH+) (HRES) MH

+ found 7752638

C30H43N6O18+

rotamers) 1459 3143 3214 3258 4359 4400 4460 4504 4574 4931 4969 5004 5757

5777 5785 5802 5829 6551 6942 6957 7011 7021 7035 16870 16891 16898 16934

16945 16957 16987 16997 17035 17083 17110 17160 17182 17275 17298 17318

95

17330 mz (ES) 733 (MH+) (HRES) MH

+ found 7333259 C30H49N6O15

tR 843 min

96

Chapter 6

61 Introduction

cell death137

α-defensin

97

β-defensin

θ-defensin

The anti-HIV-1

or

98

HO

NN

NNH

OO

O

STr STr

NHBoc NHBoc68

N

NN

N

NNO

O

OO

O

NHBoc

SH

BocHN

HS

69N

NN

N

NNO

O

OO

O

NHBoc

S

BocHN

S

70

N

NN

N

O O

O

OOO

O

SH

HS

N

NN

N

O O

O

OO

O

S

72 73

NN

OO

O

STr

71

N

O

NH

STr

HO

99

OHNN

N

NN

OO

O

OO

O

TrS

NOO

N

NN

NNH

OO

O

TrS

74N

N

N N

NN

N

NO

O

O O O

OOO

O

NO

SH

HS

N

N N

NN

N

NO

O

O O O

OOO

O

NO

S

75

76

OH

NN

N

OO

O

S

NO

O

N

NH

O

S

77

HO

NN

OO O

OO

OTrS

78

NOO

N

HN

O

O STr

N

N N

NN

N

NO

O

O O O

OOO

O

NO

HS

79

SH

N

N N

NN

N

NO

O

O O O

OOO

O

NO

S

80

S

HO

NN

OO O

OO

O

S

NOO

N

HN

O

OS

81

80 and 81

100

conditions10

bond formation145

10

621 Synthesis

and the

NH2

CH3OH Et3N

H2NNH

O

134 135O O O

O O

(Boc)2O

NH2

SHH2N

S(Ph)3COH

TFA rt quant

136 137

101

HPLCMS analysis149

N

NN

N

O O

O

OO

O

STr

TrS

139

N

NN

N

NNO

O

NHBoc

STr

BocHN

TrS

138

N

N N

NN

N

O

O O O

OO

O

N

O

NO

STr

TrS

140

N

N N

N

O

O O O

OO

O

N

O

NO

TrS

141 STr

acid151

102

in a protic solvent

61

118 7237 154

138

1

2

3

4

CuCl (40) H2O20

TFA H2O Et3SiH

(925525)17

I2 (5 eq)16

DMSO (5) DIPEA19

CH2Cl2

TFA

AcOHH2O (41)

CH3CN

-

139

5

6

7

8

9

TFA H2O Et3SiH

(925525)17

DMSO (5) DIPEA19

DMSO (5) DBU19

K2CO3 (02 M)

I2 (5 eq)154

TFA

CH3CN

THF

CH3OH

-

140

9 I2 (5 eq)154

CH3OH gt70

141

9 I2 (5 eq)154

CH3OH gt70

74

9 I2 (5 eq)154

CH3OH gt70

78

9 I2 (5 eq)154

CH3OH gt70

103

closer

63 Conclusions

641 Synthesis

Compound 135

(MH+) (HRES) MH

+ found 1891600 C9H21N2O2

+ requires 1891598

104

Compound 137

+ found 3201470

approach11

+ found 14197497

C80H107N8O11S2+ requires 14197495 100

+ found 14157912

C82H111N8O9S2+ requires 14157910 100

105

+ found 18681278

C106H155N12O13S2+ requires 18681273 100

+ found 18360650

C104H147N12O13S2+ requires 18360647 100

+ found 14017395 C80H105N8O10S2

+ requires

14017390 HPLC tR 250 min

106

+ found 13977810 C82H109N8O8S2

+ requires

13977804 HPLC tR 271 min

+ found 18501170 C106H153N12O12S2

+ requires

18501167 HPLC tR 330 min

+ found 18040390

75-76-77 and 79-80-81)

HPLCMS

107

108

FRONTESPIZIOpdf

3

Chapter 1

1 Introduction

or in diagnostics

4

11 Peptidomimetics

affinities

5

literature)6

6

is possible

7

For some

folds11

16798ndash16807

8

interactions

A B

13525ndash13530

9

helical

(SMH) presumes

10

circular peptoids20

activity22

De Riccardis26

14)

11

neoformans27

12

+ and K

+)

21a and its ability

to promote Na+H

29 However

= 1ndash5 mM)

13

identified the

and pool synthesis

14

of 2733

protein degradation

15

and temperature13

50-51 In

helix bundle

29

able to form a Zn2+

complex

16

(30-34 figure 18)21a

+ and K

17

18

Kirshenbaum

centre

19

116)

DNA recognition

Drug discovery

20

1 RNA targeting

2 DNA targeting

3 Protein targeting

4 Cellular delivery

5 Pharmacology

Nanotechnology

Pre-RNA world

21

and

ways

22

The shift

DNA hybridization

23

showed a Tm

= 227 degC

grey bonds)

Chem 2009 6113

24

workers55

Figure 125)

NH

NN

NNH

N

O O O

BBB

X n

49

O O O

11)

groups

25

Cl HON

R

O Fmoc

ON

R

O

HN

R

O

HATU or PyBOP

Cl

HOBr

O

OBr

OR-NH2

O

HN

R

O

DIC

26

NH

NOH

OO

NH2

NH

NNH

OO

Deprotection

H2NN

NH

OO

NH

NOH

OO

CouplingNH

NNH

OO

NH

N

OO

First cleavage

NH2

HNH

N

OO

B

nPNA

B-PGs B-PGs

B-PGsB-PGs

PGt PGt

PGt

27

17 Aims of the work

NH

NN

N

O

Base

OOO

NH

N

BaseO

O

N

NH

O

HO50

NH

NN

N

O

Base

OOO

NH

N

BaseO

O

N

NH

O

HO 51

FmocN

NOH

N

NH

O

t-BuO

O

n 50 n = 151 n = 5

28

and 58)

N

OO

O

N

O

N

O

56

N

NN

OO

O

N

O

57

N

O

N

O ON

N

O

O58

cyclohexapeptoid 58

HON

H

O

HON

H

O

n=661n=659n=460

n n

58

29

N

NN

N

NNO

O

OO

O

H3N

2X CF3COO-

N

NN

N

O

H3N

NH3

H3N

4X CF3COO-

62 63

N

NN

N

NNO

O

OO

O

H3N

NH3

H3N

NH3

6X CF3COO-

64

cyclohexapeptoid 64

Gd3+

were evaluated

30

NN

OOO

OH

O

HO O

HO

O

OHO

N NN

O OO OMe

NNNO

OO

MeO

NN

OOO

OH

O

OH

O

HO O

HO

O

HO

O

OHO

NN

OOO

O

OH

O

HO

O

OHO

6566

HO

NN

NNH

OO

O

STr STr

NHBoc NHBoc68

N

NN

N

NNO

O

OO

O

NHBoc

SH

BocHN

HS

69N

NN

N

NNO

O

OO

O

NHBoc

S

BocHN

S

70

59

31

N

NN

N

O O

O

OOO

O

SH

HS

N

NN

N

O O

O

OO

O

S

72 73

NN

OO

O

STr

71

N

O

NH

STr

HO

OHNN

N

NN

OO

O

OO

O

TrS

NOO

N

NN

NNH

OO

O

TrS

74N

N

N N

NN

N

NO

O

O O O

OOO

O

NO

SH

HS

N

N N

NN

N

NO

O

O O O

OOO

O

NO

S

75

76

OH

NN

N

OO

O

S

NO

O

N

NH

O

S

77

32

HO

NN

OO O

OO

OTrS

78

NOO

N

HN

O

O STr

N

N N

NN

N

NO

O

O O O

OOO

O

NO

HS

79

SH

N

N N

NN

N

NO

O

O O O

OOO

O

NO

S

80

S

HO

NN

OO O

OO

O

S

NOO

N

HN

O

OS

81

79 80 and 81

33

Chapter 2

21 Introduction

Unfortunately

and poor

cellular uptake62

N-methyl content

the oligomeric

revealed an

and

34

lipids70

(decoy)72

2001 268 6066ndash6075

FmocN

NOH

N

NH

O

t-BuO

O

n 32 n = 133 n = 5

35

221 Chemistry

intermediate 87

36

Scheme 22) The

yields

O

t-BuONH2 O

OH+

O

t-BuON

R

82 83 84 R = H

85 R = Fmoc

a

b

c

d

O

t-BuON

Fmoc

O

t-BuON

Fmoc

OHOH

OHN

O

e

O

t-BuON

Fmoc NO

OR

86 87

O

NH

O

88 R = CH3

50 R = H

f

37

O

HO

NHCbz

89

5

O

t-BuO

NH2

5

a

INHCbz

9190

b

O

t-BuO

NHCbz

5

HONH2 HO

NHCbz

92 93 94

c d

e

O

t-BuO

N

5

NHR

95 R = H R = Cbz

R

h

97 R = Fmoc R = Hg

HN

O

t-BuO

N

5

Fmoc

51 R = H

98

i

NO

OR

O

t-BuO

N

5

Fmoc

l

ON

NH

O

91 94+

99 R = CH2CH3

38

duplexa

DNA mis-matchedb

(PNA sequence)8a

486 364

(DNA sequence)9

335 265

39

23 Conclusions

--negative

241 General Methods

400 (1H at 40013 MHz

1H at 25013 MHz

13C at 6289 MHz)

CDCl3 = 770 CD2HOD

= 334 13

40

242 Chemistry

664 716 827 1728 mz (ES) 206 (MH+) (HRES) MH

+ found 2061390 C9H20NO4

+ requires

2061392

831 1201 1202 1249 1252 1273 1279 1280 1415 1438 1564 1573 1704 1713 mz (ES)

428 (MH+) (HRES) MH

+ found 4282070 C24H30NO6

+ requires 4282073

41

C (7550 MHz CDCl3) 282 473 506 508 578 584 681 686 826 827 1202 1249 1252

1273 1280 1415 1438 1439 1560 1564 1686 1687 1987 mz (ES) 396 (MH+) (HRES) MH

+

505 533 672 676 815 817 1197 1247 1249 1268 1275 1410 1437 1560 1562 1687

1694 1719 1721 mz (ES) 469 (MH+) (HRES) MH

+ found 4692341 C26H33N2O6

+ requires

4692339

Compound 88

42

475 478 482 491 506 518 521 525 530 665 682 823 825 1105 1202 1245 1248

12514 1253 1273 1274 1275 1280 1281 1413 1414 1438 1440 1511 1564 1627 1644

1691 1692 mz (ES) 634 (MH+) (HRES) MH

+ found 6342767 C34H40N4O9

+ requires 6342765

Compound 50

CDCl3) 123 282 299 464 473 487 502 509 536 683 823 826 1108 1202 1249 1252

1253 1273 1275 1280 1414 1421 1438 1440 1517 1566 1650 1652 1682 1690 1692

1723 mz (ES) 620 (MH+) (HRES) MH

+ found 6202611 C33H38N3O9

+ requires 6202608

CDCl3) 245 260 280 295 353 408 664 799 1279 1280 1283 1366 1563 1728 mz (ES)

322 (MH+) (HRES) MH

+ found 3222015 C18H28NO4

+ requires 3222018

43

+ found 1881647 C10H22NO2

+ requires 1881651

1570 mz (ES) 196 (MH+) (HRES) MH

+ found 1960970 C10H14NO3

+ requires 1960974

+ found 3059989 C10H13INO2

+ requires 3059991

44

1279 1280 1283 1361 1566 1728 mz (ES) 365 (MH+) (HRES) MH

+ found 3652437

Compound (96)

392 396 462 468 471 476 663 797 1196 1244 1268 1274 1278 1282 1364 1411

1437 1556 1563 1727 mz (ES) 587 (MH+) (HRES) MH

+ found 5873120 C35H43N2O6

+ requires

5873121

Compound (97)

351 254 388 424 465 473 479 534 670 801 1198 1199 1240 1246 1269 1271 1277

1405 1413 1438 1490 1558 1571 1727 1729 mz (ES) 453 (MH+) (HRES) MH

+ found

4532740 C27H37N2O4+ requires 4532748

Compound (98)

45

1269 1275 1413 1440 1559 1562 1722 1729 mz (ES) 539 (MH+) (HRES) MH

+ found

5393117 C31H43N2O6+ requires 5393121

Compound (99)

246 260 280 353 464 467 472 478 481 507 617 669 801 1106 1110 1199 1246

1270 1276 1413 1438 1439 1512 1564 1643 1677 1689 1730 mz (ES) 705 (MH+)

+ requires 7053500

Compound (51)

46

458 478 485 488 496 547 683 814 816 1118 1119 1212 1259 1265 1283 1290

1295 1303 1391 1426 1429 1450 1451 1526 1577 1662 1690 1727 1744 mz (ES) 677

(MH+) (HRES) MH

+ found 6773185 C36H45N4O9

+ requires 6773187

800 1730 mz (ES) 262 (MH+) (HRES) MH

+ found 2622017 C13H28NO4

+ requires 2622018 101

293 294 367 568 569 583 591 660 662 705 710 815 1746 mz (ES) 336 (MH+) (HRES)

+ requires 3362386

O

t-BuO

NH2

5

91

O

OH

83

+

O

t-BuO

NR

OHOH

101 R =OH

OH

100 R = H

5

47

ndash 283911 60

ndash

48

Chapter 3

31 Introduction

as

the

102 103

49

hexamer (figure 33)

hydrogen bonding

50

crystal lattice

networks 81-82

51

58 (figure 36)

N

OO

O

N

O

N

O

56

N

NN

OO

O

N

O

57

N

O

N

O ON

N

O

O58

cyclohexapeptoid 58

321 Chemistry

(scheme 31)

52

Cl

HOBr

O

OBr

O

HON

H

O

HON

H

O

n=6 106

n=6 104n=4 105

NH2

ONH2

n

HON

NN

O

N

O

NNH

O

N

OO

O

N

O

N

O

PyBOP DIPEA DMF

104

56

80

HON

NN

O

NH

O

N

NN

OO

O

N

O

PyBOP DIPEA DMF

105

57

columns

53

HON

NN

O

N

O

NNH

O

O O O

O

106

N

O

N

O ON

N

O

O58

PyBOP DIPEA DMF

87

peptide sequences88

4 seeding

54

precipitate

crystals

8 CHCl3

with seeding

Needlelike

crystals

precipitate

vapor phase

Crystals

55

1 CH2Cl2 Slow

evaporation

Prismatic

crystals

2 CHCl3 Slow

evaporation

Precipitate

evaporation

Crystalline

Aggregates

4 CHCl3 Hexane Slow

evaporation

Little

crystals

1 CHCl3 Slow

evaporation

Crystals

2 CHCl3 CH3CN Slow

evaporation

Precipitate

3 AcOEt CH3CN Slow

evaporation

Precipitate

5 AcOEt CH3CN Slow

evaporation

Prismatic

crystals

6 CH3CN i-PrOH Slow

evaporation

Little

crystals

7 CH3CN MeOH Slow

evaporation

Crystalline

precipitate

between two

phases

Precipitate

9 CH3CN Crystallin

precipitate

56

56A 56B

57

58

57

PM (g mol-1

) 91903 88303 58869 51336

Source Rotating

anode

Rotating

anode

Rotating

anode

Rotating

anode

λ (Aring)

154178 154178 154178 154178

a (Aring)

b (Aring)

c (Aring)

α (deg)

β (deg)

γ (deg)

4573(7)

9283(14)

2383(4)

10597(4)

9240(12)

11581(13)

11877(17)

10906(2)

10162(5)

92170(8)

10899(3)

10055(3)

27255(7)

8805(3)

11014(2)

12477(2)

7097(2)

77347(16)

8975(2)

V (Aring3 ) 9725(27) 1169(3) 29869(14) 11131(5)

Z 8 1 4 2

Dcalc (g cm-3

) 1206 1254 1309 1532

58

μ (cm-1

) 0638 0663 0692 2105

Observed

reflecti

on (Igt2I )

4883 1856 1985 1841

R1 (Igt2I) 01345 00958 00586 01165

Rw 04010 03137 02208 03972

sides

56A

59

space group is P1

inversion centre

60

arsquo 0 1 0 a b

crsquo 1 0 0 c a

structure of 56B

61

62

method (figure 315)

(a)

63

(b)

64

34 Conclusions

were reported

351 General Methods

400 (1H at 40013 MHz

1H at 25013 MHz

13C at 6289 MHz)

CDCl3 = 770 CD2HOD

= 334 13

65

352 Synthesis

approach11

+ found 9014290 C54H57N6O7

+ requires

9014289 100

+ found 6072925 C36H39N4O5

+ requires

6062920 100

+ found 7093986 C30H57N6O13

+ requires

7093984 100

66

atmosphere

+ found

8834110 C54H55N6O6+

+ found 5892740 C36H37N4O4

180 min

rotamers) 171 5 1710 1796 1701 1700 1698 1697 1695 1693 1691 1689 1687 1682

1681 717 715 714 713 710 708 706 (bs) 702 (bs) 700 (bs) 698 (bs)695 (bs) 693 (bs)

67

+ found 6913810 C30H55N6O12

Data reduction

0)(

cFFw

2

0

22

0

2

iii

iciii

Fw

FFwwR

Goumlttingen 1997

68

Rietveld analysis

56B

Data reduction

92

0

1

ii

icii

F

FFR

69

Data reduction

(154178 Aring)

Data reduction

70

Chapter 4

41 Introduction

98mdash that

efficiency

polylysine3101

) to synthetic

)

71

dendrimers113

nanoparticles114

60 452ndash472

72

108

examined a set

73

chains

N

NN

N

NNO

O

OO

O

H3N

2X CF3COO-

N

NN

N

O

H3N

NH3

H3N

4X CF3COO-

62 63

74

N

NN

N

NNO

O

OO

O

H3N

NH3

H3N

NH3

6X CF3COO-

64

cyclohexapeptoid 64

421 Synthesis

NH2

CH3OH Et3N

NH2

NH

O

110

111

O O O

O O

(Boc)2O

Cl

HOBr

O

OBr

O

NH2

NH2BocHN

111

75

HON

O

N

ONHBoc6

N

H

ONHBoc

6

2

N

H

ONHBoc

6

6HO

113

114

HON

O

N

O

N

H

ONHBoc

6

2112

HON

O

N

O

NH

ONHBoc

6

2

112

HATU DIPEA

DMF 33N

NN

N

NN

O O

O

OO

O

NHO

O

HN

O

115

76

HON

O

N

ONHBoc6

NH

ONHBoc6

2

113

N

NN

N

O

HN

NH

O

OO

O

HN

O O

116

HATU DIPEA

DMF 33

NH

ONHBoc6

6HO114

N

NN

N

NNO

O

OO

O

HNNH

HN

OO

NHO

O

NH

O

NH

O

O O

O

117

HATU DIPEA

DMF 24

77

43 Conclusions

441 Synthesis

Compound 111

+ found 2171920 C11H25N2O2

+ requires

2171916

approach11

78

+ found 11196485

C62H87N8O11+ requires 11196489 100

+ found 13378690

C70H117N10O15+ requires 13378694 100

+ found 15560910

C78H147N12O19+ requires 15560900 100

79

33 mz (ES) 1101 (MH+) (HRES) MH

+ found 11013785 C62H85N8O10

tR 206 min

conformers) 1706 1690 1689 15863 1561 1558 1557 1444 1434 1433 1408 1407 1362

1360 1279 1275 1274 1269 1264 1245 1194 817 816 675 670 660 659 598 504

500 499 487 483 467 466 390 274 205 33 mz (ES) 1319 (MH+) (HRES) MH

+ found

+ found 15380480 C78H145N12O18

225 min

1371 (bs) 1311 (bs) 1302 1297 1293 1290 1286 1283 1270 548 538 528 521 (bs) 508

3+ requires 9161797

80

+) (HRES) MH

+ found 9232792

C50H87N10O65+

requires 9232792

+ found 9433978 C48H103N12O6

7+ requires 9433970

81

Chapter 5

51 Introduction

The image

82

uncontrasted images

s-1

(20 MHz and

39degC)2

where R1

is the

1p + Ros

127

N N

NNHOOC

HOOC

COOH

(DOTA)

119

NH

N NH

N

COOH

CONHCH3H3CHNOC

HOOC

DTPA-BMA

121

N N

NNHOOC

HOOC

COOH

OH

CH3HP-DO3A

120

DTPA

NH

N NH

N

COOH

COOHHOOC

HOOC

118

83

T1M (eq 52)

52 Eq )τ(555

][

51 Eq

1

1111

MM

is

p

os

p

is

p

oobs

T

CqR

RRRR

53 Eq τω1

7τ

τω1

3τ1)S(S

r

γγ

4π

μ

15

2

T

12

c2

2

s

c2

2

c1

2

H

c1

6

GdH

2

H

2

s

22

0

1M

54 Eq τ

1

τ

1

τ

1

τ

1

EMRci i

molecules

1p and Ros

design (figure 52)1

84

NN

OOO

OH

O

HO O

HO

O

OHO

N NN

O OO OMe

NNNO

OO

MeO

NN

OOO

OH

O

OH

O

HO O

HO

O

HO

O

OHO

NN

OOO

O

OH

O

HO

O

OHO

6566

85

Na+ Ca

2+ and NH

128

of

The

synthesis131

N N NR

H

R

N

N N

R

H N

N N

R

H

R

128

Chem 2002 67 3057 131

Int Ed 2002 41 1053 132

86

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

122

531 Synthesis

O

Br -Cl+H3N O

O

OHN O

O

DIPEA DMF

18 h rt

O

Cl

O Fmoc-Cl =

HO

O

N O

O

Fmoc

123 124125

DIPEA = N

126

127 128 and 129 (figure 55)

HON

O

O Ot-Bu

H

6127

HON

O

O Ot-Bu

3N

O

H

OMe128

HON

O

O Ot-Bu

2

N

O

N

O

H

Ot-BuO

129

87

HATU DIPEA

DMF 654

NN

N

NN

OO

O

Ot-Bu

O

Ot-Bu

O

t-BuO O

t-BuO

O

t-BuO

O

Ot-BuO

HON

O

O Ot-Bu

H

6

127

130

HON

O

O Ot-Bu

3

N

O

H

OMe128

NN

N

NN

OO

O

Ot-Bu

O

t-BuO

O

Ot-BuO

HATU DIPEA

DMF 82

131

HON

O

O Ot-Bu

2

N

O

N

O

H

Ot-BuO

129

HATU DIPEA

DMF 71

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

122

88

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

NN

N

NN

OO

O

Ot-Bu

O

t-BuO O

t-BuO

O

Ot-BuO

CuSO4 5H2O

sodium ascorbate

H2OCH3OH

N NN

O O

O OMe

NNNO

OO

MeO

NO

OO

NN OMe2

53

122

133

132

afford 67

excretion

figure 57)

89

CP = cyclopeptoid

which was 315 mM-1

s-1

e 253 mM-1

s-1

)

the

134

90

2 (s

Gd-65 21times1019

275 1times10-8

280 3 15

Gd-66 28times1019

225 1times10-8

216 3 14

coordination sphere

541 Synthesis

Compound 125

91

1401 2797 3350 4430 4914 6041 8144 16888 17077 mz (ES) 232 (MH+) (HRES) MH+

Compound 126

17111 17161 17488 17504 mz (ES) 454 (MH+) (HRES) MH

+ found 454229 C26H32NO6

+

requires 454223

92

+ found 1129 6500

C54H93N6O19+ requires 11296425 80

+ found 9195248

C42H75N6O16+ requires 9195240 75

+ found 9495138

C43H73N6O15+ requires 9495134 85

93

4531 4555 4572 4594 4676 4756 4774 4858 4896 4977 5004 5037 5102 5277 5323

5340 8058 8141 16765 16776 16793 16819 16832 16856 16886 16890 16902 16915

16928 16938 16951 16993 17027 17038 17062 17069 17078 17091 17096 17114

17134 17139 17158 17167 17174 17187 65 mz (ES) 1111 (MH+) (HRES) MH

+ found

11116395 C54H91N6O18+

solution 40 mM) 2846 3478 4550 5020 8227 12724 12822 14310 17015 17173

4962 4992 5061 5397 5993 6026 7280 8195 8110 8269 11401 11800 16061 16072

17001 17075 17121 17145 17190 17219 17245 17288 17310 82 mz (ES) 901 (MH+)

3653 3671 3698 4378 4402 4421 4437 4509 4687 4755 4767 4780 4866 4903 4925

4991 5016 5053 5091 5297 5329 7236 7250 7286 8056 8127 8136 15786 15863

16720 16805 16851 16889 17026 17100 17151 17152 71 mz (ES) 931 (MH+) (HRES)

MH+ found 9315029 C46H71N6O14

94

4301 4448 4501 4564 4838 4891 4944 5060 5334 5892 6915 6999 7052 7189 8054

8069 8080 8092 8136 11387 11640 12416 12435 12446 12465 12474 12532 12547

14221 15891 15941 15952 16872 16954 17140 17055 17100 17120 17131 mz (ES) 1397

(MH+) (HRES) MH

+ found 13977780 C64H109N12O22

4531 4555 4572 4594 4676 4756 4774 4858 4896 4977 5004 5037 5102 5277 5323

5340 8058 8141 16765 16776 16793 16819 16832 16856 16886 16890 16902 16915

16928 16938 16951 16993 17027 17038 17062 17069 17078 17091 17096 17114

17134 17139 17158 17167 17174 17187 mz (ES) 775 (MH+) (HRES) MH

+ found 7752638

C30H43N6O18+

rotamers) 1459 3143 3214 3258 4359 4400 4460 4504 4574 4931 4969 5004 5757

5777 5785 5802 5829 6551 6942 6957 7011 7021 7035 16870 16891 16898 16934

16945 16957 16987 16997 17035 17083 17110 17160 17182 17275 17298 17318

95

17330 mz (ES) 733 (MH+) (HRES) MH

+ found 7333259 C30H49N6O15

tR 843 min

96

Chapter 6

61 Introduction

cell death137

α-defensin

97

β-defensin

θ-defensin

The anti-HIV-1

or

139 a)

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